To conclude, the new morphometric image analysis method we developed, using a routine and cheap staining, may provide valuable tool to assess IF and thus to evaluate new sources of grafts.Early interstitial fibrosis (IF) correlates with long-term renal graft dysfunction, highlighting the need for accurate quantification of IF. However, the currently used Banff classification exhibits some limitations. The aim of our study was to precisely describe the progression of IF after renal transplantation using a new morphometric image analysis method relying of Sirius Red staining. The morphometric analysis we developed showed high inter-observer and intra-observer reproducibility, with ICC [95% IC] of respectively 0.75 [0.67–0.81] (n = 151) and 0.88 [0.72–0.95] (n = 21). We used this method to assess IF (mIF) during the first year after the kidney transplantation from 66 uncontrolled donors after circulatory death (uDCD). Both mIF and interstitial fibrosis (ci) according to the Banff classification significantly increased the first three months after transplantation. From M3 to M12, mIF significantly increased whereas Banff classification failed to highlight increase of ci. Moreover, mIF at M12 ( Kidney transplantation remains the optimal treatment for CKD, offering a better survival than dialysis and being cost-effectiveness13. Moreover, as DBD kidney transplantation, DCD kidney transplantation is associated with increased survival of patients who have end-stage renal disease (ESRD) and are on the transplant waiting list14. Whereas early reports of uDCD showed good renal outcome and no increase in IF/TA compared to extended criteria DBD, other reported a very early and more severe development of IF/TA15 than in DBD.In order to increase the pool of available donors in a setting of organ shortage, grafts from uncontrolled donors after circulatory death (uDCD) have been used in France since 2006. Several studies have shown that kidneys from DCD provide almost an equal function as kidneys from donation after brain death (DBD)20. CAD corresponds to the irreversible replacement of functional renal tissue by extracellular matrix (ECM) proteins, leading to the progressive impairment of renal graft function. Among the pathological lesions observed during CAD, one of the most prominent is interstitial fibrosis and tubular atrophy (IF/TA). Other histological damages include glomerulosclerosis, splitting of glomerular capillary basement membranes and vascular intimal hyperplasia21. Many mechanisms which are of immunological origins or not, are involved in this multifactorial and complex process26. The current concept states that many processes leading to graft fibrosis, i.e. IF/TA, occur early after the transplantation, especially within the first few months27. IF/TA involves about 40% of kidney grafts at 3–6 months and up to 50% at 1 year, while renal function remains stable29, which suggests that renal function is a suboptimal and late marker of kidney graft dysfunction. Up to now, the only reliable method to assess IF/TA is the histological evaluation of a graft biopsy31 which also allows to determine specific lesions and pathogenic processes affecting the graft23. It is also well-known that early IF correlates with long-term graft dysfunction34, highlighting the need for accurate quantification of IF to better identify patients requiring specific therapeutic interventions and to determine the efficacity of such interventions. IF in kidney graft is usually graded using the Banff classification35. However, the Banff classification exhibits some limitations. The semi-quantitative evaluation in only 4 grades (0 to 3) prevents the precise evaluation of IF evolution and may be not sensitive enough in the early stages. Furthermore, studies have shown a wide interobserver variation in the assessment of renal graft biopsies using the Banff classification38.Whatever the donor’s status, chronic allograft dysfunction (CAD), which is the final result of different etiological and pathogenetic conditions, remains the first cause of graft lossThe aim of the study was to assess the initial progression of interstitial fibrosis and tubular atrophy in kidney grafts from uDCD formerly classified as “Maastricht II” non heart beating donors, using a new image analysis method based on Sirius Red staining.In the first part of our work, we will evaluate the accuracy, the robustness and reproducibility of our computerized analysis method (mIF) to assess IF. In the second part, mIF will be applied to assess IF during the first year after kidney transplantation from uDCD and mIF score will be correlated with clinico-biological data.39 or because the paraffin block was worn out. Measurement of interstitial fibrosis by image analysis (mIF) was performed on the 151 biopsies independently by two pathologists (CP and SF)     between the four groups defined by the Banff classification. Mean mIF (%) was respectively 8.3 ± 2.4 (ranges: 4.3–15.8) in the ci0 group (n = 40), 10.7 ± 3.6 (ranges: 4.0–25.2) in the ci1 group (n = 82), 17.1 ± 6.4 (ranges: 9.8–36.6) in the ci2 group (n = 18) and 20.1 ± 8.0 (ranges: 5.7–32.7) in the ci3 group (n = 11). The correlation between mIF and Banff ci was 0.62 with 95% CI of [0.51–0.71] (p < 0.001). Discrepancies between ci and mIF were observed in only 2 cases. These cases were carefully reanalyzed: interstitial edema and inflammation were the main drawback to accurately quantify ci in Massons’ trichrome staining  according to the Banff criteriaSF) Fig. . The val29) Fig. . To asse-8) Fig. . Analysi25) Fig. . Then, w25) Fig. . The mIFThe 66 patients who received kidney graft from uDCD included 14 females and 52 males. Mean age at the time of kidney transplantation was 44.7 ± 9.8 years old . Indications for kidney transplantation were hypertensive nephroangiosclerosis (n = 16), IgA nephropathy (n = 7), autosomal dominant polycystic kidney disease (n = 6), Alport syndrome (n = 4), diabetic nephropathy (n = 3), other glomerulopathies (n = 5), tubulo-interstitial nephritis (n = 3) and uropathy (n = 4). Nephropathy remained undetermined in 18 patients. All patients received induction therapy with anti-lymphocyte serum. They also received mycophenolate mofetil, corticosteroids and tacrolimus per local practice. Characteristics of the recipient patients are summarized in Table in situ preservation was 145.9 ± 15.2 minutes . Mean cold ischemia was 16.2 ± 3.7 hours . Delayed graft function occurred in 54/66 patients (82%). Mean time between transplantation and renal graft recovery was 22.6 ± 9.8 days . Non-primary graft function occurred in 2 patients. Biopsy-proven acute rejection occurred in eight patients (12%). Mean LDH level reached a pic at Day 3 post transplantation (2539 ± 1089 IU/L) which was significantly associated with time to graft recovery .The 66 patients received kidney graft from 48 uDCD Table . Donors -3) and M12 . Conversely, chronic lesions, especially interstitial fibrosis and tubular atrophy, significantly increased over time. According to the Banff staging system, interstitial fibrosis remained unchanged from D0 to D15/30 , increased from D0 to M3  and remained stable between M3 and M12   before organ donation. Thereafter, 20 patients underwent kidney graft biopsy between 15 and 30 days (D15–30) post-transplantation for delayed graft function, 28 at 3 months (M3) and at 12 months (M12) for routine evaluation. Socio-demographic and clinical data were not significantly different between the patients who underwent biopsies at D15–30, M3 and M12. Histological features of biopsies are summarized in Table 37) Fig. .Table 2Hp < 10−3). Conversely to ci according to the Banff scoring system, increase of mIF remained significant from M3 to M12 (p = 0.021). Fibrosis tended to increase as early as the first month after renal transplant (p = 0.056). Most of the patients underwent more than one kidney biopsy. Among them, seven patients underwent early kidney graft biopsy between D7 and D30 and protocol biopsies at M3 and at M12  but not at D0, D15–30 and M3 correlated with mean time to renal graft function recovery. In addition, mIF at M12 correlated with increase of creatininemia at M12  and at last follow-up . We found the same correlation using the Banff scoring system, however, whereas there was no correlation between ci according to the Banff scoring system at M3 and creatininemia at M12 , mIF at M3 tended to be associated with increase of creatininemia at M12 .Baseline characteristics of uDCD , tWIT and cold ischemia were not associated with mIF at any time. mIF at M12 48 compared to polarized Sirius Red and trichrome staining, but also significant correlation with the decrease of GFR33.The aim of this study was to precisely describe the evolution of IF after renal transplantation with uDCD, using a new computerized image analysis method. Indeed, IF is one of the most prominent pathological lesions of CAD and correlated with renal graft prognosisIn our uDCD cohort, both mIF and ci according to the Banff classification significantly increased the first three months after renal transplantation From M3 to M12, mIF significantly increased whereas Banff classification failed to highlight increase of ci. Then, mIF may be more accurate than semiquantitative Banff evaluation to identify the effects of antifibrotic therapeutic interventions. Morphometric IF tended to increase in the first month after renal transplantation but without reaching significance, due to the limited size of sample, graft biopsies being not systematically performed during the first month. However, this early and sensitive quantification of IF may be a very valuable surrogate marker to study therapeutic intervention aiming the early development if CAD in kidney transplantation. Moreover, whereas increase of creatininemia at M12 was not associated with ci according to the Banff scoring system at M3, it tended to be associated with mIF at M3, suggesting usefulness of a more sensitive quantification analysis.In our study, mIF at M12 was significantly associated with increase of creatininemia at M12 and at last follow-up, which confirms the need for a precise assessment of IF.53. Surprisingly, few studies have focused on morphometric quantification of IF on native kidneys55. Hunter et al.54 demonstrated than high collagen matrix index and fibrillary collagen index, both assessed by quantitative morphometry after Sirius Red staining, predicted relapse and progression to ESRD during lupus nephritis. Similarly, Gibyeli Genek et al.55 performed a quantitative evaluation of IF with Sirius Red in IgA nephritis and highlighted that such evaluation might serve as an effective novel method to determine the prognosis in IgA nephritis.IF, which corresponds to the replacement of renal functional tissue by ECM, is not only a major concern after kidney transplantation, but also during CKD in native kidneys. Indeed, renal fibrosis still represents the final target to treat CKDTo conclude, the new morphometric image analysis method we developed, using a routine and cheap staining, may provide valuable tool to assess IF during chronic allograft dysfunction and thus to evaluate new sources of grafts. The use of this method to describe CKD on native kidney should also be evaluated.After institutional review board, we retrospectively included all the 66 patients who received a kidney graft from uDCD between 2007 and 2012 in Bicêtre hospital. Kidney donation followed the 2008 Declaration of Istanbul principles and the French Agence Nationale de la Biomédecine regulation. Research was approved by the committee of the Centre de Ressources Biologiques . Informed written consent was given by all the patients for the scientific use of the graft biopsies in the CRB, Paris Sud University. All procedures and the use of tissues were performed in accordance with the Declaration of Helsinki principles. Clinical reports and biological data were collected from the associated clinical database.56. Biopsy samples were fixed in formalin, acetic acid and alcohol (AFA), paraffin-embedded and sliced 2.5 µm thick. Slides were stained with HES , Masson’s trichrome (3 sections), periodic acid Schiff, Jones methenamine silver and Sirius red. Third Masson’s trichrome and Sirius red staining were consecutive. Kidney graft biopsies were reviewed independently by 2 pathologists (CP and SF) for histological features according to Banff recommendations35 in a blind manner from clinical data. Immunostaining for C4d was performed using a rabbit monoclonal A24-T anti-human C4d antibody  and a Leica BOND-MAX™ autostainer . Epitope retrieval was achieved using the ready-to-use Bond Epitope Retrieval Solution 1  after 30 min heating.A total of 166 kidney graft biopsies was performed in the 66 patients. The biopsies were processed for routine light microscopy, as we previously describedRenal biopsy sections sliced 2.5 µm sliced and stained with Sirius red were digitalized by a ScanScope Aperio scanner (CS), using 20X objective. For each biopsy, the cortical section, defined as the part inside the renal capsule and outside the medulla, was manually selected on digital slides. Glomeruli and medium-sized arteries were deleted by the operator.57. Briefly, the operator manually selects internal Sirius Red negative areas and positive areas and next runs the software that show the final selection of the Red area. The internal negative control step allows the comparison of slides that are sequentially stained in various batches of Sirius Red since staining usually may vary. Finally, the result was expressed as a percentage of the red positive area on the total cortical surface. Morphometric analyses were performed twice for each biopsy in an independant manner by two pathologists (CP and SF) who had no knowledge of the clinical data.Renal-cortex fibrosis was quantified using a computer-based morphometric analysis software  as previously publishedt tests for continuous variables, Fisher’s exact and chi-squared tests for categorical variables were performed. The intraclass correlation coefficient (ICC) with its 95% CI was used to study the reproducibility of morphometric IF (mIF) measures. Correlations between quantitative variables were assessed with Pearson product-moment correlation coefficient. For all analyses, a p value <0.05 was regarded as significant. Analyses were performed using R software (version 3.2.0)59, InStat 3 software  and Prism 4 (GraphPad Software).Descriptive statistical methods  were used to assess the distributions of variables. Wilcoxon rank sum and Supplementary Information.


Hypertensive disorders of pregnancy (HDP), such as preeclampsia (PE) or the Hemolysis Elevated Liver enzymes and Low Platelets (HELLP) syndrome are associated with elevated cardiovascular disease (CVD) risks, but standardized prevention guidelines after such pregnancies are lacking. Hypertension is the first emerging risk factor after PE/HELLP pregnancies and is a major risk factor for CVD. Hypertension before the age of 55 years may lead to various manifestations of end-organ damage at relatively young age. Therefore, timely treatment of elevated blood pressure is mandatory, but many of these high-risk women have long-term undetected and untreated hypertension before adequate treatment is initiated.The aim of our study is to assess whether home blood pressure monitoring (HBPM) in women with a previous PE/HELLP pregnancy is a valuable tool for the early detection of hypertension.Women with a history of both early and late PE/HELLP syndrome aged 40–60 years are invited to participate. Patients with a history of CVD, known hypertension and/or use of antihypertensive medication are excluded. Women are randomized between HPBM or ‘usual care’. The primary outcome is feasibility and usability of HBPM after 1 year of follow-up.Secondary outcomes will be the effectiveness of HPBM to detect hypertension, the efficacy of BP treatment, quality of life, health-related symptoms, work ability, and life-style behaviour.The results of this study will provide better strategies for timely detection and prevention of hypertension in women after PE/HELLP.ClinicalTrials.gov, NCT03228082. Registered June 15, 2017. Cardiovascular disease (CVD) is the leading cause of mortality in women worldwide. One of the major risk factors is high blood pressure . In womeHDP complicate 10–15% of all pregnancies , 5. ThisPE and HELLP syndrome represent the most severe HDP with the highest CVD risk and complicate 3–5% of all pregnancies –8. PE isMany women with a history of PE/HELLP remain undiagnosed for their hypertension, since female-specific risk factors are not routinely accounted for in the SCORE or other cardiovascular risk charts. These young women leading busy lives are not likely to seek medical attention to check their blood pressure regularly, and they do not want to be considered as ‘patients’ lifelong either. Therefore, elevated blood pressure may remain undetected and untreated for many years. These considerations can be surpassed using self-monitoring applications.The aim of our study is to assess whether home blood pressure monitoring (HBPM) in women with a previous history of PE/HELLP is a valuable tool in clinical practice for early detection and timely treatment of hypertension.The etiology of PE is still largely unknown. It is hypothesized that PE/HELLP is a result of poor placentation causing a widespread inflammatory response resulting in generalized endothelial dysfunction in the placental and maternal circulation , 12. A bEvidence is increasing that maternal endothelial dysfunction persists after such a complicated pregnancy, increasing the risk of hypertension and end-organ damage at young age , 18. FurIn the last two decades, a large body of evidence has shown that women after PE/HELLP are at increased risk to develop hypertension before the age of 55 years. In a meta-analysis by Bellamy et al., a relative risk of 3.70  2.70–5.05) was found for hypertension after PE . We showUp to half of an individual’s CVD risk is attributable to high blood pressure, which makes timely diagnosis of hypertension very important . In the HBPM is a simple way to check an individual’s usual blood pressure and has better reproducibility than ABPM . SeveralUsing HBPM, hypertension is diagnosed if the average blood pressure of twice-daily measurements for at least 3, but preferably 7, days is ≥135/85 mmHg , 29, 31.The feasibility and usability of HBPM to detect hypertension will be evaluated in a multicenter randomized controlled trial (RCT). Participants are women aged 40 to 60 years with a history of PE/HELLP. Seven departments of Obstetrics in the Euregio Rijn-Waal, in The Netherlands and Germany, participate as recruiting centers. All study visits will be executed at the department of Cardiology at the Radboudumc, the principal study center. The databases of the seven departments of Obstetrics were searched for patients with PE and/or HELLP in their medical history. Previous PE/HELLP was defined as diastolic blood pressure (BP) ≥ 90 mmHg and/or systolic BP ≥ 140 mmHg and proteinuria ≥300 mg/24 h, more than 1 year ago. Both patients with early-onset PE (i.e. delivery before 34 weeks of gestation) and late-onset PE (i.e. delivery from 34 weeks of gestation onwards) will be included, as well as patients after eclampsia or HELLP syndrome. Exclusion criteria are previous cardiovascular events , already diagnosed hypertension and/or use of antihypertensive medication, not having a smartphone, or inability to perform self-measurement of blood pressure. Pregnant women or women aiming to have another pregnancy will be excluded. All eligible patients are invited to participate by letter. In case no response is received after two weeks, we will call the patient to ask for participation. The participating women are randomized to HBPM (intervention group) or ‘usual care’ (control group) using the randomization method in Castor EDC . PatientAt the baseline visit, the randomization and a physical examination will be conducted consisting of measurements of blood pressure, heart rate, weight, height, and waist and hip circumference. Standardized blood pressure measurements will be performed on both arms, with at least 3 different readings. Waist and hip circumference will be measured with a non-stretchable tape measure over light clothing: waist circumference at the narrowest point of the abdomen and hip circumference at the widest diameter around the buttocks. Finally, a non-fasting blood sample (13 mL) will be drawn. The blood samples will be centrifuged at 1500G for 15 min within 4 h, separated into equal volumes (250 μL), and frozen immediately at minus 80 °C. These samples will be stored for future biomarker analysis.All participants will also be asked to complete structured online questionnaires at several occasions during follow-up. At baseline, information will be obtained on personal medical history, family history of CVD, obstetric history, cardiovascular symptoms, menopausal symptoms, and lifestyle . In addition, several standardized questionnaires will be administered, such as the 36-item Short Form Health Survey (SF-36), the Copenhagen Psychosocial Questionnaire (COPSOQ-II) on working ability  and workHBPM will be performed using the Withings BPM device. This device was validated for home blood pressure measurements according to the ESH International Protocol . The WitThe primary outcome is the feasibility and usability of HBPM during 1 year of follow-up. The participants in the intervention group will receive monthly feedback on their blood pressure measurements. In case of elevated blood pressure, participants will be advised to contact their general practitioner for treatment advice. Feasibility and usability as well as protocol adherence and persistence to HBPM will be determined using the questionnaires completed after 6 months and 1 year of follow-up and the actual BP measurements. Protocol adherence is the proportion of participants in the intervention group who self-monitor their blood pressure twice daily for at least 4 consecutive days every month during 1 year of follow-up. Persistence to HBPM is defined as the proportion of participants in the intervention group who measure their blood pressure until the final follow-up visit. The secondary outcomes include blood pressure levels and the prevalence of hypertension in the intervention group compared to the control group at 1 year of follow-up. In addition, quality of life, work capability, and cardiac and/or hypertensive symptoms will be compared between the groups.Formally, a sample size calculation is not required for a feasibility study. The prevalence of PE/HELLP in the Dutch/German population is 3–5% of all pregnancies per year. Among 5.5 million inhabitants in the study region as a whole, we expect approximately 55,000 pregnancies each year, among which an estimated 2200 are complicated by PE/HELLP. If the participating obstetric centers  cover approximately 20% of these pregnancies, more than 4000 women with PE/HELLP should have been seen in the past 10 years. These women can be considered our study base from which the study participants can be recruited.Within the time-frame and the financial constraints of the study, it would be realistic to recruit approximately 200 eligible women, with inclusion of 100 women in the intervention group and 100 women in the usual care group. For the secondary endpoint BP, we would be able to find differences of approximately 5 or more mmHg in SBP (assuming a SD of 12 mmHg) and 4 or more mmHg in DBP (with a SD of 9 mmHg) with 80% power and a 0.05 level of significance (two-sided), assuming less than 10% loss-to-follow-up. In comparison to other self-monitoring of blood pressure studies, a difference of 5 mmHg in systolic blood pressure is plausible .Data analysis will be performed using SPSS (IBM). The characteristics of the participants will be described using means with 95% confidence intervals for normally distributed continuous variables or medians plus ranges otherwise, and proportions for categorical variables. Both qualitative and quantitative analyses will be performed on the primary and secondary outcomes with a range of statistical techniques to compare the baseline data with the 6 months and 1 year follow-up data taking into account study group. To describe protocol adherence and persistence to HBPM, survival analyses will be performed.NCT03228082, https://clinicaltrials.gov/ct2/show/NCT03228082, date of registration: June 15, 2017.This study was approved by the Regional Committee on Research involving Human Subjects Arnhem-Nijmegen (number 2016–3006). This trial was registered in the Clinical Trials Register, We aim to demonstrate that HBPM is an acceptable and easy way to check blood pressure regularly. We assume that by using HBPM, the blood pressure in de intervention group will be regulated better after 1 year of follow-up compared to the control group. By including the patient actively in HBPM, we expect to be able to diagnose hypertension earlier and to establish more timely treatment. If HBPM shows to be a feasible method to diagnose hypertension in the high-risk women included in this study, this method may be useful for follow-up starting directly after pregnancies complicated by hypertensive disorders. The incidence of hypertension among women with these pregnancy complications is expected to be even higher than the findings of this study will show, since we exclude women with already diagnosed hypertension. A limitation is that we cannot confirm the diagnosis hypertension using ABPM for all women in the study. However, recent studies showed a diagnostic agreement ranging from 80 to 90% between the two methods . Therefo


Acute respiratory distress syndrome (ARDS) can be a devastating condition in children with cancer and alveolar recruitment maneuvers (ARMs) can theoretically improve oxygenation and survival. The study aimed to assess the feasibility of ARMs in critically ill children with cancer and ARDS.20, with a delta pressure of 15 cmH2O for 60 seconds. We assessed blood gases pre- and post-maneuvers, as well as ventilation parameters, vital signs, hemoglobin, clinical signs of pulmonary bleeding, and radiological signs of barotrauma. Pre- and post-values were compared by the Wilcoxon test. We retrospectively analyzed 31 maneuvers in a series of 12 patients (median age of 8.9 years) with solid tumors (n=4), lymphomas (n=2), acute lymphoblastic leukemia (n=2), and acute myeloid leukemia (n=4). Patients received positive end-expiratory pressure from 25 up to 40 cmH3. Post-maneuvers, mean arterial pressure decreased more than 20% in two patients, and four needed an increase in vasoactive drugs. Hemoglobin levels remained stable 24 hours after ARMs, and signs of pneumothorax, pneumomediastinum, or subcutaneous emphysema were absent. Fraction of inspired oxygen decreased significantly after ARMs . Oxygen partial pressure (PaO2)/FiO2 ratio increased significantly (p=0.0002), and the oxygenation index was reduced (p=0.01), but all these improvements were transient. Recruited patients’ 28-day mortality was 58%. Median platelet count was 53,200/mm ARMs, although feasible in the context of thrombocytopenia, lead only to transient improvements, and can cause significant hemodynamic instability. Acute respiratory failure is a frequent condition in children with cancer admitted to the Intensive Care Unit (ICU). Those who develop acute respiratory distress syndrome (ARDS) and need mechanical ventilation (MV) have a poor prognosis.Few studies have analyzed alveolar recruitment in the pediatric population and none within the cancer subpopulation. The present study aimed to evaluate the feasibility of ARMs in critically ill pediatric cancer patients who developed acute respiratory failure due to ARDS.,This is a retrospective study approved by the Ethics Committee of Universidade Federal de São Paulo (UNIFESP) (protocol no. 12802/2009), and the informed consent form was waived. We evaluated all data on electronic medical records from patients aged 0 to 17 years, admitted to our 11-bed oncologic ICU from January 1, 2010 to December 31, 2011, with an ARDS diagnosis according to the American-European Consensus Conference.2O. The initial PEEP value varied from 25 to 40 cmH2O. It was kept for 60 seconds if no hemodynamic instability  was observed. ARMs could be performed three times if oxygenation was not sustained. For example, children with less than 20 kg were submitted to an initial ARM at a PEEP of 25 cmH2O. ARM could be repeated at a PEEP of 30, with one last ARM at a PEEP of 35 cmH2O. Children with more than 20 kg were submitted to the first ARM at a PEEP of 30 cmH2O, the second (if necessary) at a PEEP of 35, and the last one at a PEEP of 40, always with a delta pressure of 15 cmH2O.Following an institutional protocol, all patients were under continuous sedation and received neuromuscular blockade (rocuronium) before ARMs. Patients had to be at the first week of disease. ARMs were carried out in pressure-controlled ventilation mode, with a delta pressure  of 15 cmH2O above this desaturation point  × mean airway pressure (mPaw) / oxygen partial pressure in arterial blood (PaO2).We analyzed blood gases before and after ARMs, as well as MV parameters, heart rate, mean arterial pressure (MAP), pulse oximetry, and vasopressor doses. Chest radiographs were reviewed to detect pneumothorax, pneumomediastinum, and pulmonary interstitial emphysema. Hemoglobin levels and subcutaneous emphysema were also monitored for 24 hours after ARMs. Oxygenation index (OI) was calculated according to Ortiz et al.: OI = fraction of inspired oxygen .3 .We analyzed 31 maneuvers performed in 12 patients, with a median age of 8.9 years (ranging from 9 months to 16 years). 2O [interquartile range (IQR) 27-75: 35-40 cmH2O]. Maximum PIP was 55 cmH2O (IQR: 50-55 cmH2O), and the mean respiratory rate was 4.7 breaths per minute [standard deviation (SD): 3].During ARMs, the minimum PEEP was 25 and the maximum, 40 cmHWe found no significant changes in heart rate during ARM . During seven maneuvers performed in five patients, MAP had a decline greater than 5% , leading to an increase in rates of infusion of vasoactive drugs in four of them (33%). In two patients (16.6%), the reduction in MAP was greater than 20%. During six ARMs performed in two patients (16.6%), we identified a transient increase in MAP greater than 5% . Mean MAP was 79 mmHg (SD: 16.8) before and 80.2 mmHg (SD: 18.5) after alveolar recruitment (p=0.93).None of the children experienced a decrease in hemoglobin levels in the 24 h following ARMs. No major air leaks were detected, such as pneumothorax, pneumomediastinum, and/or subcutaneous emphysema.2 and OI significantly decreased after ARMs ; the PaO2/FiO2 ratio had a statistically significant increase (p=0.0002). These improvements in oxygenation were not sustained for more than 2 hours after ARMs, as oxygen saturation declined, and FiO2 increased again. All other blood gas parameters analyzed  showed no signs of significant improvement post-maneuvers. Dynamic lung compliance was not affected either.FiOPEEP levels remained high after ARMs (p=0.02). Other ventilatory parameters  showed no significant differences. 2 pre- and post-maneuvers  and also in PaO2/FiO2 ratios pre- and post-maneuvers . Unfortunately, we could not perform further analyses of these first ARMs due to the small sample size.We also analyzed the first ARM of each patient. Considering only these 12 ARMs, we found significant differences in FiOIn our small group of critically ill patients, recruitment maneuvers produced some degree of improvement in oxygenation parameters, but these improvements were evanescent. The fact that one-third of patients needed more vasoactive drugs is also concerning. The literature has no other studies on ARMs in this population, so we cannot compare results.Pediatric cancer patients who develop ARDS are extremely ill and have high mortality rates. Ben-Abraham et al. studied 17 children with ARDS and hematologic malignancies under MV; 11 of them died (64.7%).,,2/FiO2 ratio that could last up to 12 hours after the maneuvers. In our study, however, improvement lasted for no more than 2 hours, showing the transient efficacy of ARMs, also registered by Kheir et al.Recruitment maneuvers can improve hypoxemia, but their use in critically ill children with cancer causes concerns due to frequent thrombocytopenia and the risk of pulmonary hemorrhage. The best method for performing ARMs is yet to be defined.No significant changes were found regarding the patients’ heart rates. Cruces et al. pointed out hypotension as a common side effect of ARM, detecting MAP reductions of 9.2%.As ARMs improve oxygen saturation during a short period, healthcare providers might feel tempted to perform them frequently, but no data available from controlled studies allow strong conclusions on their efficacy, safety, and long-term consequences in children.The main limitation of our study is having a sample too small to determine all aspects of ARM safety. Nonetheless, it was performed as a pilot study to assess the feasibility of ARMs in these extremely ill patients.2, and not as a routine therapy in ICUs.A recent large, well-designed, adult clinical trial resulted not only in limited success but in higher 28-day mortality in the ARM group;In conclusion, ARMs, although viable, have restrictions regarding their effectiveness in children with cancer and ARDS, with transient improvements in oxygenation, but no improvements in lung compliance. Despite the lack of bleeding-related complications, even in the context of thrombocytopenia, hemodynamic instability seems to be a major concern.


UV light enhances nitric oxide availability by mobilizing storage forms in the skin, suggesting incident solar UV radiation may lower blood pressure. We tested this hypothesis by exploring the association between environmental UV exposure and systolic blood pressure (SBP) in a large cohort of chronic hemodialysis patients in whom SBP is determined regularly.Hypertension remains a leading global cause for premature death and disease. Most treatment guidelines emphasize the importance of risk factors, but not all are known, modifiable, or easily avoided. Population blood pressure correlates with latitude and is lower in summer than winter. Seasonal variations in sunlight exposure account for these differences, with temperature believed to be the main contributor. Recent research indicates that UV radiation and temperature data for each clinic location were retrieved from the National Oceanic and Atmospheric Administration database. Linear mixed effects models with adjustment for ambient temperature, sex/age, body mass index, serum Na+/K+ and other covariates were fitted to each location and combined estimates of associations calculated using the DerSimonian and Laird procedure. Pre‐dialysis SBP varied by season and was ≈4 mm Hg higher in black patients. Temperature, UVA and UVB were all linearly and inversely associated with SBP. This relationship remained statistically significant after correcting for temperature.We studied 342 457 patients  at 2178 US dialysis centers over 3 years. Incident UV radiation is associated with lower SBP. This raises the possibility that insufficient sunlight is a new risk factor for hypertension, perhaps even in the general population.In hemodialysis patients, in addition to environmental temperature, incident solar  In a large North American cohort of chronic hemodialysis patients, incident solar ultraviolet radiation was found to be associated with lower systolic blood pressure, even after adjustment for environmental temperature, showing that human exposure to ultraviolet light contributes to blood pressure regulation and accounts for seasonal and geographical variation.While this is an observational study, our findings are consistent with data showing reduced blood pressure and lower rates of cardiovascular disease in populations with higher sun exposure, and suggest that Vitamin D independent benefits of sunlight should be considered when assessing risk benefit ratios.Sunlight exposure appears to lower blood pressure; insufficient exposure to natural ultraviolet radiation and/or active avoidance of sunlight may be new risk factors for hypertension.Hypertension is a leading global cause for premature disease and death, affecting >1 billion people worldwide. Its pathophysiology involves complex interactions of multiple genetic, behavioral/lifestyle‐related and environmental risk factors and social stressors with the endocrine, metabolic, autonomic nervous and cardiovascular systems. Without overt symptoms, hypertension can remain unrecognized and escape detectionThe 2017 ACC/AHA GuidelinesSeasonal BP variation was first reported in ischemic heart disease patients in the early 1960s.While epidemiological data suggest a role for sunlight in lowering BP, its mechanism of action is uncertain. The warmth of sunshine may contribute to but cannot account entirely for seasonal BP variation when measured in a temperature‐controlled doctor's office or at home. In 2010, we proposed that solar ultraviolet (UV) radiation may contribute to cardiovascular health by releasing nitric oxide (NO) from storage forms in the skinThis observational study included 342 457 patients undergoing chronic hemodialysis in 2178 US Fresenius Medical Care facilities  at each wavelength from 280 to 400 nm using the tropospheric UV and visible radiation model from the US National Center for Atmospheric Research (NCAR) (http://cprm.acom.ucar.edu/Models/TUV/Interactive_TUV/), corresponding to >25 million hourly records for UVA and UVB for the entire observation period. We then computed hourly UVA and UVB as the summations of spectral irradiances over wavelength ranges 321 to 400 and 280 to 320 nm, respectively. Lastly, we computed summations of hourly UVA and UVB over each day to approximate the total exposure, and averages of daily UVA and UVB to calculate monthly averages. See Figures Our goal was to investigate associations between SBP and UV, with adjustment for clinical covariates and ambient temperature. Since it was not feasible to determine personal exposures to UV radiation and temperature, we approximated these exposures using environmental data retrieved from public databases at matched locations. The 2178 facilities were divided into 1530 geographical locations according to their zip codes. For each location, we first computed hourly spectral irradiances (W/mDaily average temperatures (°C) for all locations were derived from the US National Oceanic and Atmospheric Administration web site (http://www.ncdc.noaa.gov/cdo‐web/search). See Figure P values. To assess the assumption of the second stage analysis, we fitted thin‐plate splines to explore spatial patterns. We considered 3 analyses for associations between SBP and UVA/UVB: Model 1: no adjustment for covariates; Model 2: adjustments for sex, age, hypertension, use of central venous catheter as vascular access, body mass index, interdialytic weight gain, albumin, erythropoietin dose, hemoglobin, serum sodium, and potassium, and a linear trend over calendar time; Model 3: adjustment for ambient temperature in addition to all Model 2 covariates. As expected, incident UV radiation and environmental temperature were closely associated. To detect potential problems with collinearity, we computed the variance inflation factor of temperature when UVA or UVB was in the model. The variance inflation factors equaled 2.82 for the combination of temperature and UVA and 2.85 for temperature and UVB; since both were smaller than the cutoff value of 5, we deemed that collinearity was not a problem. Separate analyses were performed for black and white patients. We used the complete case analysis since we have a large data set, from which there was only a small percentage of missing values  We first fitted a linear mixed‐effects model for repeated measurements from all patients at each location with an unstructured covariance matrix for random intercept and slope. This model takes into account between‐patient variability and the within‐patient correlation of SBP measurements across time points. This approach produced estimates of population intercept and slope at each location. (2) We then computed a combined estimate of association based on estimated population slopes from all locations using the DerSimonian and Laird procedure for random effects meta‐analysis,es Table . Other tes Table .Our cohort included a large fraction of US hemodialysis patients undergoing in‐center hemodialysis between January 2011 and December 2013. Over a third of patients were black, consistent with the high incidence of end‐stage renal disease in this community, and the average follow‐up time was ≈12 months  W/m2, respectively. Although UV and temperature were closely associated (correlation coefficients between UVA/UVB and temperature: 0.80/0.81), collinearity was deemed not to be a problem.By computing monthly averages from a total of 45 784 963 individual BP measurements recorded over 36 months we observed clear seasonal variation in SBP Figure A. IndepeWe next fitted 3 separate models to adjust for possible confounders. The SBP fall with increasing incident UV exposure was seen in Model 1 (unadjusted data) and Model 2 (with adjustment for covariates). Correcting for temperature (in Model 3) did not alter the statistically significant inverse relationship between UV and SBP, albeit less strongly than before , the predominant form of UV reaching the earth's surface, will partly pass through window glass and penetrate as deep as the dermis in skin. Although not absorbed by DNA (and thus not directly mutagenic), it is absorbed by other photosensitizers and consequently generates free radicals, producing oxidative stress. UVB (280–320 nm) is responsible for the photochemical synthesis of previtamin D3 from 7‐dehydrocholesterol, but is also a direct DNA mutagen. It makes up ≈5% of incident UV radiation, and is around 2 orders of magnitude more erythemogenic than UVA. Shorter wavelength UVB penetrates less well through the atmosphere, so is reduced more than UVA at low altitude and as the sun's elevation falls in winter at higher latitudes. Individuals with high measured vitamin D levels have lower BP, but meta‐analyses of vitamin D supplementation studies,0 nm, theWeThe duration of action of the BP‐lowering effects of UV exposure is uncertain. Experimentally, a single exposure to UV lowers blood pressure for <1 hour in health volunteers,Irrespective of mechanism, mean SBP was higher and the age younger in black than white patients, reflecting the well‐documented higher incidence of hypertension and faster trajectory of renal function decline in blacks than whites.We suggested earlier that exposure to natural sunlightThe present study has strengths and limitations. Its strengths relate to the large and diverse patient population, managed according to a standard protocol, by a single large dialysis provider with measurements recorded by professional healthcare providers, allied to high‐quality National Center for Atmospheric Research/National Oceanic and Atmospheric Administration environmental data. Weaknesses include the lack of personal‐level UV exposure measurements, differences in BP monitoring devices, the absence of data on anti‐hypertensive therapy, BP in‐between dialysis visits, socioeconomic status and physical activity level of patients. Our conscious decision to omit diastolic BP from our analyses represents another study limitation. We caution that the data relate to a North‐American cohort and may not necessarily be relevant for other geographical locations and populations of distinct genetic heritage. We have mitigated the absence of individual data on anti‐hypertensive medication by including the diagnosis of hypertension as a covariate, as it is a proxy marker for medication. This would reduce the apparent effect size of UV. While circulating vitamin D levels have been used to estimate personal exposure to UV radiation in other studies this approach would not be suitable in hemodialysis patients many of whom receive cholecalciferol supplementation to prevent secondary hyperparathyroidismSeasonal variation in blood pressure has been known for 40 years, but, to our knowledge, for the first time we show here that this occurs independently of temperature. The reduction in blood pressure is more marked with a rise in UVB than UVA, and in whites than black people. Dermatological concerns about the skin cancer inducing effects of UV radiation need to be balanced against the observed blood pressure lowering effects of sunlight, particularly given the greatly higher burden of disease caused by hypertension.We acknowledge support from the US National Science Foundation (DMS‐1507620), the Center for Scientific Computing at the California NanoSystems Institute, the UK Medical Research Council (G1001536) and the Faculty of Medicine, University of Southampton.Drs Maddux, Usvyat, and Kotanko hold stock in Fresenius Medical Care. Dr Kotanko receives author honoraria from UpToDate. Prof Feelisch and Dr Weller are members of the Scientific Advisory Board of AOBiome, LLC and Relaxsol, Ltd. The remaining authors have no disclosures to report.Data S1.Figure S5. Seasonal variation in  systolic blood pressure superimposed onto linear downward trend in African‐American and white hemodialysis patients.Table S1. Percentages of All Variables With Missing DataTable S2. Relationship Between Systolic Blood Pressure and Ultraviolet Light Stratified by Race and Temperature and Longitude Based on Model 1Table S3. Relationship Between Systolic Blood Pressure and Ultraviolet Light Stratified by Race and Longitude Based on Model 2Table S4. Relationship Between Systolic Blood Pressure and Ultraviolet Light Stratified by Race and Longitude Based on Model 3Table S5. Relationship Between Systolic Blood Pressure and Ultraviolet Light Stratified by Race and Latitude Based on Model 1Table S6. Relationship Between Systolic Blood Pressure and Ultraviolet Light Stratified by Race and Latitude Based on Model 2Table S7. Relationship Between Systolic Blood Pressure and Ultraviolet Light Stratified by Race and Latitude Based on Model 3Table S8. Estimated Coefficients From Model 2, Stratified by UV Wavelength Band and RaceTable S9. Estimated Coefficients From Model 3, Stratified by Wavelength Band and RaceTable S10. Estimated Coefficients From Model 2 With an Extra Term of AGE2, Stratified by Wavelength Band and RaceTable S11. Estimated Coefficients From Model 3 With an Extra Term of AGE2, Stratified by Wavelength Band and RaceTable S12. Relationship Between Systolic Blood Pressure (SBP) and Standardized Ultraviolet Light as Well as Standardized Temperature, Stratified by Race and Wavelength BandTable S13. Relationship Between Systolic Blood Pressure (SBP) and Ultraviolet Light, Stratified by Race and TemperatureFigure S1. Geographical location of dialysis facilities at which blood pressure data were collected (A) and spatial distribution of patient numbers by State (B).Figure S2. Average UVA (W/m2) in the 4 seasons.Figure S3. Average UVB (W/m2) in the 4 seasons.Figure S4. Average temperature (°C) in the 4 seasons.Click here for additional data file.


Gastrointestinal stromal tumor (GIST) initiation and evolution is commonly framed by KIT/PDGFRA oncogenic activation, and in later stages by the polyclonal expansion of resistant subpopulations harboring KIT secondary mutations after the onset of imatinib resistance. Thus, circulating tumor (ct)DNA determination is expected to be an informative non-invasive dynamic biomarker in GIST patients.We performed amplicon-based next-generation sequencing (NGS) across 60 clinically relevant genes in 37 plasma samples from 18 GIST patients collected prospectively. ctDNA alterations were compared with NGS of matched tumor tissue samples (obtained either simultaneously or at the time of diagnosis) and cross-validated with droplet digital PCR (ddPCR).We were able to identify cfDNA mutations in five out of 18 patients had detectable in at least one timepoint. Overall, NGS sensitivity for detection of cell-free (cf)DNA mutations in plasma was 28.6%, showing high concordance with ddPCR confirmation. We found that GIST had relatively low ctDNA shedding, and mutations were at low allele frequencies. ctDNA was detected only in GIST patients with advanced disease after imatinib failure, predicting tumor dynamics in serial monitoring. KIT secondary mutations were the only mechanism of resistance found across 10 imatinib-resistant GIST patients progressing to sunitinib or regorafenib.ctDNA evaluation with amplicon-based NGS detects KIT primary and secondary mutations in metastatic GIST patients, particularly after imatinib progression. GIST exhibits low ctDNA shedding, but ctDNA monitoring, when positive, reflects tumor dynamics. KIT juxtamembrane domain, encoded by exon 11. Similar complexity is found in other KIT regions  [Gastrointestinal stromal tumor (GIST) is a rare cancer of mesenchymal origin, with an incidence rate of approximately 1 case/100,000/year . Oncogen and 17) . Likewis and 17) . Althoug and 17) –10 that  and 17) , 8–10. RImportantly, KIT/PDGFRA primary and secondary genotype is relevant for GIST clinical management because it predicts GIST clinical behavior and efficacy from tyrosine kinase inhibitors (TKIs) with KIT inhibitory activity in the first line  – imatinqPCR or digital PCR-based technologies have the highest analytical sensitivity for mutation detection –20. WhilOverall, there is an urgent need for real-time tumor biomarkers to guide therapy selection in GIST. Nevertheless, until ctDNA is proven to render the genomic information detected in solid tissue, it cannot replace the need for metastatic tissue biopsy of patients, nor guide clinical decisions . To addrLocalized and metastatic KIT- or PDGFRA-mutant GIST patients were prospectively enrolled in a tissue and plasma acquisition protocol. Consenting patients were distributed in two cohorts: cohort A (matched tissue/plasma) included localized or metastatic GIST patients with matched tissue and plasma samples obtained simultaneously. Localized patients were imatinib-naïve and tissue samples were obtained through surgical removal of the primary tumors. Tumor tissue in metastatic patients included resections of unifocal progressive disease. In all cases plasma samples were collected 7 to 14 days before tumor resection. Plasma samples from patients on TKI treatment were obtained while on drug. Cohort B  included metastatic patients with serial plasma samples throughout the course of their treatment, together with tumor tissue at the time of diagnosis.This study was approved by the Institutional Review Board from each participating center and written informed consent was obtained from all patients to donate blood samples and tumor tissue.Peripheral blood was collected into EDTA tubes (Beckton Dickinson) and plasma was extracted within 4 h of blood collection through two centrifugation steps of 10 min each, the first at 1600 g and the second at 3000 g. Single-use 1.5 mL plasma aliquots were obtained and stored at − 80 °C until use. cfDNA was obtained from 3 mL of plasma using the QIAamp Circulating Nucleic Acids kit (Qiagen) and quantified with a Qubit Fluorometer (ThermoFisher Scientific).Representative formalin fixed, paraffin-embedded (FFPE) tumor tissue blocks were retrieved from each case and reviewed by a GIST expert pathologist (S.L.). Five 10-μm tissue sections with more than 20% tumor area were obtained. DNA extraction was performed with the automated system Maxwell16 FFPE plus LEV DNA purification kit (Promega). DNA quality and concentration were measured with a NanoDrop 1000 spectrophotometer .KIT exon 11 long insertions and/or deletions (indels) (> 15 base pairs) through Sanger Sequencing [In order to optimize VHIO amplicon-sequencing pipeline, seven additional FFPE primary tumor samples were retrieved from our GIST series database with known quencing .KIT exon 11 in-frame deletions was also used to optimize the NGS pipeline for the detection of long indels (> 15 base pairs). GIST-T1 has deleted 19 aminoacids (KIT exon 11 p.V560_Y578del), and GIST430 has 17 (KIT exon 11 p.V560_ L576del) [DNA from two human GIST cell lines with known long An initial multiplex-PCR with a proof-reading polymerase was performed on all samples. Tumor and plasma DNA were sequenced with an in-house developed amplicon-sequencing panel of over 1330 primer pairs targeting frequent mutations in oncogenes and several tumor suppressors, totaling 60 genes  and the maximum occurrences for extending a long deletion (−d), and decreasing the penalties for opening (−O) or extending a gap (−E). The resulting SAM file was used as input for the indel caller SOAPindel.Recent studies revealed shortcomings of state-of-the-art variant callers that might fail to detect complex indels . For thiThe QX200 ddPCR System  was used to confirm in plasma the presence of variants detected in tumor tissue and plasma by amplicon sequencing. Genomic DNA from tumor tissue from the same patient was run as positive control. Custom Taqman SNP genotyping assays for ddPCR were designed to detect KIT and PDGFRA mutations , 900 nM of each primer, 250 nM of each probe and 8 μL of cfDNA or 30 ng per reaction in FFPE (positive controls). Each assay was performed in triplicate in separate mixes and loaded in different wells for amplification. The thermal cycling program was performed according to specifications of the manufacturer. After PCR, droplets were read in the Droplet Reader and analyzed with QuantaSoft version 1.7.4. Human reference genomic DNA was included as negative control and used to determine the cutoff for allele calling in each assay. ddPCR validations were carried out blinded to tumor genotype information.Descriptive statistics were used to characterize patients at study entry. Fisher’s exact test, Mantel-Haenzel test and Mann-Whitney test were used, depending on each variable type, to determine the association between clinicopathological and molecular features with detection of ctDNA. Concordance of VAFs between plasma ddPCR and NGS was calculated using Pearson Correlation. All statistical tests were conducted at a 2-sided significance level of 0.05. Analyses were performed using GraphPad Prism 6  or IBM SPSS Statistics 20.0 .n = 5) or metastatic (n = 8) patients with matched tissue and plasma samples collected simultaneously. Matched tissue in metastatic patients included resections of a single progressing lesion (n = 5) or surgery of unifocal progressive disease aiming R0 disease (n = 3). Cohort B  included nine imatinib-resistant, metastatic GIST patients treated with sunitinib or regorafenib in which serial plasma samples were collected simultaneously to CT-scans throughout the clinical course of the disease. These nine patients include four from cohort A that also had serial plasma determinations. Patients in cohort B had tumor tissue available, either at diagnosis or through tumor resection of metastatic disease (cohort A).The current study recruited prospectively 18 KIT- or PDGFRA-mutant GIST patients from July 2015 to December 2017. Figure Clinicopathological and molecular features from all GIST patients included in this study are shown in Table KIT exon 11 was evaluated. A total of nine cases, including two GIST cell lines, with known KIT exon 11 genotypes, were used to validate the alternative pipeline for the detection of large (> 15 base pairs) and/or complex indels in KIT exon 11.An alternative pipeline for the detection of large indels in KIT exon 11  or progressing to first-to-third line TKIs at the time of tissue and plasma collection , D820Y (patient 10) and D816V (patient 11)  and PDGFRA exon 12 (patient 17) were detected across the four plasma samples. KIT secondary mutations were found in the two KIT-driven GIST patients, affecting exclusively KIT exon 17 at the codons D816V in patient 11, and N822K and Y823D in patient 18.Cohort B consists of nine metastatic, imatinib-resistant GIST patients with serial plasma determinations, totaling 28 timepoints  . All but one mutation was confirmed by ddPCR. As expected, ddPCR had better sensitivity for plasma mutations than NGS, 42.9% (9/21) . Conversely, only GIST patients with advanced disease were more prone to have ctDNA detected in plasma, as evidenced by higher tumor burden (p = 0.005), involvement of several anatomic sites (p = 0.009), and having received several lines of TKIs (p = 0.003)  was not associated with ctDNA detection . Detection rate increases in metastatic patients , and particularly at the time of active/progression disease in imatinib-resistant patients. The highly-sensitive ddPCR technology raised the ratio of ctDNA detection, being also capable to detect known cfDNA mutations in one patient at the time of imatinib progression, and in a small proportion of cases with indolent disease (responsive to treatment)  led to disease stabilization together with suppression of primary KIT exon 11 and secondary KIT exon 17 Y823D mutations to undetectable levels for more than 1 year. Disease progression occurred 2 months after a necessary dose reduction, accompanied by re-emergence of KIT exon 17 Y823D resistant mutation and the appearance of a new KIT exon 17 N822K clone, likely due to insufficient dose of regorafenib against KIT secondary mutations. Detection of these mutations was largely confirmed by respective ddPCR assays. Remarkably, a slight increase in the AF of the primary KIT exon 11 mutation (1%) and the secondary KIT exon 17 Y823D  was observed by ddPCR on the third timepoint (10.2016) before radiological tumor progression, thereby anticipating in 2 months the eventual disease progression. Together, detection and monitoring of primary and secondary resistance mutations in ctDNA appears to provide dynamic information in a subset of imatinib-resistant GIST patients.From nine metastatic, imatinib-resistant GIST patients treated with sunitinib or regorafenib and with serial plasma determinations , both NGS and ddPCR found cfDNA mutations in four patients 44%), whereas ddPCR detected ctDNA in three NGS-silent patients. Two patients were NGS- and ddPCR-silent  and at low AF  compared to the majority of neoplasms –24, 42. We found amplicon sequencing of ctDNA informative in a subset of GIST, mainly in metastatic, progressive disease after imatinib failure. Therefore it has the potential to avoid tumor biopsies when tumor genotyping is required. Additionally, serial ctDNA assessment reproduces the course of the disease and provides information on subclonal dynamics. Notably, we confirmed that monitoring of known KIT or PDGFRA mutations in plasma with ddPCR is useful in a bigger subset of GIST patients, and that might predict tumor progression before radiological evaluation. Nonetheless, NGS of plasma advantages digital PCR-based technologies in the detection of the higher variety of mutations found in GIST. This is non-trivial, since KIT secondary genotype predicts response to TKIs after imatinib failure –17, 30, Unlike prior reports, an important focus of our studies was on imatinib-resistance disease, with 11 out of 18 patients in this setting. We did not identify substantial heterogeneity of KIT secondary mutations neither in plasma nor in tumor tissue, which agrees with previous PCR-based studies , 16, 44 The main limitation from our study is the cohort size: despite exhaustive inter-platform analysis and cross-validations, these low numbers cannot capture the biological complexity of this disease from paucisymptomatic localized tumors to TKI-refractory disease. This limitation, also affecting prior publications in GIST and sarcomas , 45–47, Novel ultra-deep NGS assays for plasma sequencing have the potential to detect a wider array of mutations at lower AFs, and therefore, to provide more thorough information regarding monitoring and determination of resistance mechanisms. However, the aforementioned challenges with variants at low AF (< 1%) are yet to be technically addressed in the forthcoming years , 40, 41,Amplicon-based NGS robustly detects cfDNA mutations in a subset of GIST patients, mostly restricted to imatinib-resistance, progressive and bulky disease. Although ddPCR is more sensitive than NGS, overall, ctDNA levels in GIST appear to be lower in GIST than in other neoplasms, which limits its clinical use.Additional file 1: Table S1. Genes covered by VHIO amplicon-sequencing panel.Additional file 2: Table S2. Primers and sequences for ddPCR.Additional file 3: Table S3. Known KIT exon 11 long/complex indels called with two different pipelines.Additional file 4: Table S4. Correlation of KIT/PDGFRA genotype between tissue and plasma.Additional file 5: Table S5. Association between clinicopathological factors and the presence of ctDNA in plasma.Additional file 6: Figure S6. Concordance of allele frequency between NGS and ddPCR for detection of plasma mutations in all plasma samples studied with both assays (A). ctDNA detection and allele frequencies distributed by samples detected only by ddPCR, NGS or both technologies.Additional file 7: Figure S7. Hematoxylin & eosin and c-KIT immunohistochemical stains for GIST cases 8 , 12  and 10  at baseline  and at the time of tumor progression , showing loss of c-KIT expression in the absence of resistance mutations.Additional file 8: Figure S8. Schematic view of the resistance mechanisms found in our series by NGS of plasma and tissue, and histological evaluation. Mechanisms of resistance are grouped according to its determination by ctDNA evaluation or by tumor tissue sequencing or histological evaluation.


Expression of hsa-miR-30a-5p was correlated with hypertension. We conclude that CKD patients show an increased risk of CSA-AKI and mortality after cardiovascular surgery, associated with the expansion of the CD14++CD16+ subset of proinflammatory monocytes and with IL1β expression. We propose that inflammation associated with CKD may contribute to atherosclerosis (ATH) pathogenesis.Cardiovascular mortality increases with decreasing renal function although the cause is yet unknown. Here, we have investigated whether low chronic inflammation in chronic kidney diseases (CKD) could contribute to increased risk for coronary artery diseases (CAD). Thus, a prospective case–control study was conducted in patients with CAD and CKD undergoing coronary artery bypass graft surgery with the aim of detecting differences in cardiovascular outcomes, epicardial adipose tissue volume, and inflammatory marker activity associated with renal dysfunction. Expression of membrane CD14 and CD16, inflammatory cytokines and chemokines, mitogen-activated protein (MAP) kinases and hsa-miR-30a-5p were analyzed in peripheral blood mononuclear cells (PBMCs). Epicardial fat volume and tissue inflammation in perivascular adipose tissue and in the aorta were also studied. In the present study, 151 patients were included, 110 with CAD (51 with CKD) and 41 nonCAD controls (15 with CKD). CKD increased the risk of cardiac surgery–associated acute kidney injury (CSA-AKI) as well as the 30-day mortality after cardiac surgery. Higher counts of CD14 Atherosclerotic vascular disease is more common and severe in patients with chronic kidney disease (CKD) and increases their mortality risk ,2. Howev++/CD16−, “intermediates” CD14+CD16++, and “non-classical” CD14++/CD16+ monocytes  and intermediate [CD14++/CD16+] monocytes. Finally, we subdivided CD16+ monocytes into functionally distinct intermediates CD14++CD16+ and non-classical CD14+CD16+ cells using a rectangular gate and aligning the base of the intermediates with the bottom of the concentric circles within the classical population [Monocyte subpopulations were identify using an inclusion gating strategy . Brieflypulation . Gates wTM PBMC Isolation Tubes, StemCell technologies, Vancouver, BC, Canada). Samples from aorta and perivascular adipose tissue were extracted during the intervention when it was feasible. Total RNA was extracted using the Maxwell RSC miRNA Tissue KIT  and cDNA templates synthesized with the TaqMan® Advanced miRNA cDNA Synthesis Kit . All real-time quantitative PCRs were performed by triplicate on a 7900HT Fast Real-Time PCR Instrument  and using TaqMan® Fast Advanced Master Mix. Expression analysis was performed using the comparative CT (ΔΔCT) method using β-actin as an endogenous control for normalization. Hsa-miR-30a-5p expression was normalized using hsa-miR-16-3p as an endogenous reference gene and 100 pmol/mL of exogenous cel-miR-54.PBMCs were isolated less than 2 h after blood collection by Ficoll fractioning .Continuous variables are presented as means (SD). The proportion of deaths in surgical patients with CKD was analyzed with a chi-square test. To check the association of intermediate CD14We have identified biomarkers linking CKD and CAD that might improve the current understanding of how an impaired kidney function leads to higher cardiovascular risks, as well as help to identify novel therapeutic targets. Our data suggest that only patients with upregulation of IL1β would get benefit from anti-IL1β therapy.CKD is a risk factor for CSA-AKI and death after cardiac surgery.++CD16+ monocytes, and IL1β) and could be useful to stratify the cardiovascular risk of patients with CKD.CKD patients with CAD present a proinflammatory environment that can be detected in the blood (increased C-reactive protein, fibrinogen levels, intermediate CD14We suggest a link between microvascular network density and inflammation since we observed a link between has-miR30a-5p and IL1β expression.Key points of our study:


The receiver operating characteristic curves drawn from the multiple logistic regression model comprising these five variables had an area under the curve (AUC) of 0.701 . Examination of sensitivity, specificity, and likelihood ratios (LRs) for these five variables for clinical utility showed a slightly higher sensitivity for age ≥50 years (0.849) and respiratory rate ≥18 bpm (0.769); higher specificity for systolic blood pressure ≤100 mmHg (0.938), pulse rate ≥100 bpm (0.834), and respiratory rate ≥26 bpm (0.887); higher positive LR for systolic blood pressure ≤100 mmHg (2.039) and pulse rate ≥110 bpm (2.729); and slightly lower negative LR for age ≥50 years (0.656), male sex (0.647), respiratory rate ≥20 bpm (0.669). These results are meaningful as they quantify the intuition of a skilled clinician, which can help in clinical decision making, reduce errors, and promote clinical education. Our study provides a basis for explaining to novice healthcare providers that the careful observation of ED patients, even in the absence of special laboratory tests, can help them to make judgments regarding the disposition of the patients from the ED. In conclusion, age, sex, systolic blood pressure, heart rate, and respiratory rate were independently associated with a disposition from the ED. A multivariate model including these five variables showed the moderate-quality potential to predict admission from the ED. The sensitivity, specificity, and LR of systolic blood pressure, heart rate, and respiratory rate showed the characteristics of each vital sign. These provide healthcare providers in the ED an immediate clue regarding the patient’s illness.The emergency department (ED) is a complex and busy environment that requires rapid decision making. We assessed the relationship between disposition from the ED and information that can be obtained at a glance in the ED. The presentation of the implications of commonplace information could assist healthcare providers in ensuring smooth and safe ED care. Thus, we aimed to quantitatively assess how readily obtainable findings, such as age, sex, and vital signs, are involved in the disposition of adult patients transferred to the ED. This retrospective observational study was conducted in the ED of a regional university hospital containing approximately 600 beds. Of the 685 patients included in the analysis, 351 patients were admitted to the hospital (including 12 deaths in the ED) and 334 patients were discharged from the ED. A multiple logistic regression model that included age, sex, systolic blood pressure, heart rate, respiration rate, temperature, and SpO In the emergency department (ED), decisions are made rapidly in a complex and busy environment. Decisions regarding a patient’s disposition are especially difficult in this setting. Several attempts have been made to predict admission from the ED; however, there is a trade-off between simplicity and accuracy . To imprIn contrast, this study assessed the relationship between disposition from the ED and information that can be obtained at a glance in the ED. The presentation of the implications of commonplace information could assist healthcare providers in ensuring smooth and safe ED care. In addition, this information is educational as it reveals important points to consider when examining ED patients.Thus, the current study aimed to quantitatively assess how readily obtainable findings, such as age, sex, and vital signs, are involved in the disposition of patients transferred to the ED.Study setting and participantsThis retrospective observational study was conducted in the ED of a regional university hospital with approximately 600 beds. Our ED is an advanced medical facility that receives approximately 1600 ambulances per year.The eligibility criteria were patients aged >18 years who were transported to the ED. We analyzed a database in which the age, sex, and vital signs were recorded on the arrival of patients to the ED. The analysis included data from July 1, 2018 to February 28, 2019. The Ethics Committee of Akita University School of Medicine approved this retrospective study . The need for informed consent was waived due to the observational nature of the study.Outcome and variablesThe outcome of this study was the admission from the ED. All patients who were transferred to a general ward or the intensive care unit (ICU) and those who died in the ED were considered cases of admission from the ED. Patients who were discharged from the ED were not considered.The database included information on the method of arrival (ambulance or helicopter), age, sex, systolic blood pressure, diastolic blood pressure, respiratory rate, heart rate, temperature, blood oxygen saturation, and disposition of patients transported to the ED. The vital signs in the database were measured at the time of arrival in the ED. The disposition included discharge from the ED, admission to the general ward, admission to the ICU, and death in the ED. All the information contained in the database was included in the analysis. The first choice of vital sign collection methods are the non-invasive measurement of the blood pressure at the upper extremity, anterior thoracic three-point monitor electrocardiogram to determine the heart rate, axillary temperature for body temperature, visual measurement of respiratory rate by the ED nurse, and percutaneous oxygen saturation (SpO2) for oxygen saturation. The database did not contain diagnostic information, treatment information, or laboratory data. Patient age and sex were automatically retrieved from the electronic medical records; other information was manually recorded by the ED nurse at each patient visit.Statistical methodsDifferences between cases of admission from ED and discharge from ED were assessed using the Wilcoxon rank-sum test for continuous variables and the chi-square test for categorical variables. Multivariate logistic regression analyses were performed by adjusted for age, sex, and vital signs. Since the systolic and diastolic blood pressures were correlated , only systolic blood pressure was included. A receiver operating characteristic (ROC) curve was drawn from the model using variables that were significant in the previous multivariate logistic analysis and the area under the ROC curve (AUC) was calculated to examine the fit in predicting hospitalization from the ED.The sensitivity (Se), specificity (Sp), positive likelihood ratio (LR+), and negative likelihood ratio (LR-) for the outcome were calculated for the variables that were significant in the multivariate logistic analysis. The cutoff values for each variable were examined while conducting sensitivity analysis among the abnormal values. Age was analyzed every five years from 50 to 80 years, hypotension was analyzed every 10 mmHg from 80 to 110 mmHg for systolic blood pressure, hypertension was analyzed every 10 mmHg from 140 to 220 mmHg for systolic blood pressure, pulse rate was analyzed every 10 bpm from 80 to 120 bpm, and respiratory rate was analyzed every 2 bpm from 18 to 28 bpm. Missing data were not imputed.Statistical analyses were conducted using Stata® software, version 15.1 . Significance was defined as a two-sided p-value of <0.05.2.Of the 1,059 eligible patients, patients for whom vital signs were not recorded were excluded from the analysis. These patients included those who used an ambulance to transfer to our ward from another hospital but who were not treated in the ED, those who had data entry errors in nurses’ database, and those admitted to the ED for cardiopulmonary arrest. Of the 685 patients who were included in the analysis, 351 patients were admitted to the hospital (including 12 deaths in the ED) and 334 patients were discharged from the ED , sex (p<0.001), systolic blood pressure (p=0.023), heart rate (p<0.001), and respiratory rate (p=0.028) and hospitalization from the ED . Compared to previous studies, the current study used the simplest variables from the essential information available when a patient is transferred to the ED. The variables we have adopted have the advantage of easy availability, with only blood pressure measurement requiring special medical equipment. Our variables are nonspecific and, therefore, comparatively less sophisticated in terms of accuracy. Paker et al. proposed a model for predicting hospitalization from the ED that considered age, race, place of residence, visit the day of the week, visit time, Singapore triage category , method of arriving at the hospital, and fever status, which showed an AUC of 0.825 [. CompareFurthermore, we evaluated the Se, Sp, and LRs for the five variables independently associated with admission from the ED  to quantify their clinical utility. Regarding age, the Se was high for age ≥50 years Se 0.849), suggesting that patients aged <50 years were less likely to be admitted from the ED. Furthermore, older age did not necessarily mean a higher tendency for admission from the ED. Regarding systolic blood pressure, the Sp increased from ≤100 mmHg (Sp 0.938) and was more marked with a lower cutoff. The same trend was observed for LR+, increasing for ≤100 mmHg (LR+ 2.039). In patients with systolic blood pressure ≤100 mmHg, healthcare providers should be aware of the potential for the patient to be admitted from the ED. Regarding heart rate, the Sp increased from ≥100 bpm (Sp 0.834) and further increased at higher cutoffs. The same trend was observed for LR+, increasing from ≥110 bpm (LR+ 2.729). A heart rate of ≥120 bpm showed an Sp of 0.978 and an LR+ of 4.891, strongly suggesting admission from the ED. Similar to systolic blood pressure, a pulse rate of 100 was also a warning sign. Thus, a clinical decision to discharge a patient with a heart rate ≥120 bpm from the ED should be re-evaluated. The respiratory rate tended to slightly differ from the other vital signs. A respiratory rate of ≥18 bpm showed a relatively, slightly high Se of 0.769 and a respiratory rate of ≥20 bpm showed a relatively low LR- of 0.669. Neither systolic blood pressure nor heart rate had any meaningful Se or LR-; therefore, the implications of respiratory rate differ from those of other vital signs. This may reflect the finding that tachypnea is observed in almost all conditions of clinical decline before the detection of abnormalities in other vital signs [49, suggeOur study is meaningful as it provides a key assessment of the moment a healthcare provider encounters a patient in the ED. In particular, our study provides a basis for explaining to novice healthcare providers that the careful observation of ED patients, even in the absence of special laboratory tests, can help them to make judgments regarding the disposition of the patients from the ED.This study has several limitations. First, because patient populations and indications for admission vary between hospitals, the external validity of our findings requires testing in EDs of different hospitals. Second, our results have moderate predictive quality for clinical application in isolation. However, in the real-world setting, clinicians combine symptoms, physical findings, laboratory data, and social context to determine patient dispositions; hence, our results can assist clinicians. Third, our database does not include information on pre-existing diseases, treatment, diagnosis, or length of stay in the ED, because this study was designed to evaluate essential patient information that can be obtained at a glance in ED practice. However, additional patient information could detail the characteristics of the ED practices in the study and provide further insight related to patient dispositions. In the future, we intend to build a database that includes information such as treatment and diagnosis to improve the quality of the study. Finally, vital signs may have missing values and contain recorder bias; for instance, the data may not have been recorded because the patient was clearly very ill or not ill.In conclusion, age, sex, systolic blood pressure, heart rate, and respiratory rate were independently associated with the disposition from the ED. A multivariate model including these five variables showed the moderate-quality potential to predict admission from the ED. The Se, Sp, and LR of systolic blood pressure, heart rate, and respiratory rate showed the characteristics of each vital sign. These provide healthcare providers in the ED an immediate clue as to the patient’s illness.


Smoking remains a powerful risk factor for death in end-stage renal disease (ESRD) and so is the presence of fluid overload. The relationship between smoking, blood pressure (BP) control and volume overload is insufficiently explored in patients on maintenance dialysis.This is a retrospective cross-sectional cohort study, utilizing existing patients’ data generated during routine ESRD care, including bimonthly protocol bioimpedance fluid assessment of the volume status.p: .002); nonsmokers took an average 0.73 ± 0.92 antihypertensive medications vs. 1.73 ± 1.21 for smokers (p: .0001). The distribution of taking more antihypertensive medications is skewed toward a higher number among the smokers (2x5 chi square p: .004). By bioimpedance spectroscopy, nonsmokers had an average 10.93 ± 7.65 percent overhydration (OH) over the extracellular space compared to 17.63 ± 8.98 in smokers (p: .005).We analyzed the data of 63 prevalent patients receiving thrice weekly maintenance hemodiafiltration treatments at one rural dialysis unit in Hungary. The cohort’s mean ± SD age was 61.5 ± 15.3 years; 65% male, 38% diabetic, with a mean arterial blood pressure (MAP) 99.5 ± 16.8 mmHg and Charlson score 3.79 ± 2.04. Of these, 38 patients were nonsmokers and 25 smokers. The nonsmokers’ MAP was 94.3 ± 14.0 versus smokers’ 105.9 ± 18.9 mmHg (Smoking may be a significant mediator of not only BP but also of chronic fluid overload in ESRD patents. Additional, larger studies are needed to explore the mechanistic link between smoking and volume overload. The cardiovascular mortality rate of dialysis patients is about 10 to 20 times higher than that of the general population based on USRDS data . DialysiSmoking seems to exert its deleterious effects through the acceleration of atherosclerotic disease. It is highly controversial whether hypertension as defined by high pre-HD blood pressure readings is an independent risk factor for mortality in HD patients – in fact, low pre-HD may be associated with higher mortality. The contribution of hypertension to the observed high risk in cardiovascular mortality in dialysis patients being uncertain, it is logical to look for additional mechanisms. In this study, we set out to investigate possible relationships between smoking and overhydration, an established independent predictor of increased cardiovascular mortality in this population. We devised a data review to see if smoking simply results in vasoconstriction or perhaps another mechanism may contribute to the development of hypertension and increased mortality.This is a cross sectional study of existing clinical data obtained through the clinical practice at a for-profit chain dialysis center in a rural environment in Hungary where the overall prevalence of smoking was 28% in 2014 [We reviewed the data of 63 prevalent chronic patients receiving in-center on-line hemodiafiltration. All patients who had been dialyzed at our facility for at least 2 months were included in the study, with the only exception of those with missing demographic, bioimpedance or smoking status data. We used pre-dialysis BP’s for comparison of mean arterial BP’s of the cohorts and mid-week values for bioimpedance results. Mean arterial pressure (MAP) was calculated as MAP = 1/3 (SBP – DBP) + DBP.t test to detect significance between the various parameters involving continuous data and we used Chi square test for categorical data. Multivariate regression analysis was also utilized when the associations determining fluid status and blood pressure  were examined. Statistics were calculated using GraphPad 5.04  and figures were graphed using the same. Multivariate regression analysis was calculated by a professional mathematician to predict outcome of multiple categories using IBM SPSS . We set the p value for significance to less than .05.We used p: .002); systolic/diastolic pressures 146/68 ± 24/151 for nonsmokers 160/80 ± 15/73 mmHg for smokers . p: .0001). The distribution of antihypertensive medications taken is skewed toward a higher number among the smokers (2 × 5 chi square p: .004). While 50% of nonsmokers took no antihypertensive medications at all, this rate was only 19% among the smokers. At the same time, only 7.9% (3 of the 38) nonsmokers took 3 antihypertensive medications and none took 4; the smokers’ proportion of taking 3 antihypertensive medication was 30.8% (8 of 25) and one patient was even on 4 antihypertensive medications in this group (p: .005) (Patients’ demographic data are displayed in is group . Most imp: .005) .p: .001) for smoking. As shown in p: .001) while all other above-mentioned independent variables’ multivariate coefficients were not statistically significant. For the MAP only age had a statistically significant (<.0001) bearing with a coefficient of −0.605 but not smoking. For absolute lean tissue mass (LTM) smoking had not an influence and for the relative LTM the multivariate coefficient was not significant at 6.076 (p: .087). For the relative LTM% the only significant (p: .035) coefficient was for the diabetic status −7.747. For absolute fat mass the multivariate regression coefficient was of −13.321 (p: .005) and this significance remained present for percent fat mass at −6.506 (p: .02). Though these issues regarding fat and lean tissue mass were not the subject of our investigation, there may be a well-recognized association between smoking and fat mass. r: 0.54 p: <.0001) correlation with their fluid overload [Smoking causes blood pressure elevation by several mechanisms including an impairment of endothelium-dependent vasodilation  and endooverload . Patientoverload . Thus, ioverload  have alsoverload  to have p values less well below .01 makes us confident of the validity of the study. This is an observational, retrospective study, not a trial; therefore a cause and effect association could not be stated. We still feel, however, that these observations, namely that smoking may be associated with fluid overload are clinically important findings. We conclude that smoker dialysis patients’ blood pressure may be more volume-related than that of nonsmokers in addition to the other deleterious effects of smoking.While it is admittedly the weakness of the study that the total number of dialysis patients is relatively low, the statistical strength of our findings with