Lymphoma involving the central nervous system and CNS relapse present diagnostic and predictive challenges. Its diagnosis is based on conventional methods with low sensitivity and/or specificity. More powerful tools for its early detection, response evaluation, and CNS relapse prediction are needed. MicroRNAs are short post-transcriptional gene regulators that are remarkably stable and detectable extracellularly in body fluids. We evaluated the diagnostic and predictive potential of circulating oncogenic microRNAs (oncomiRs) in CSF and plasma for the detection of secondary CNS involvement in aggressive B-NHL lymphomas, as well as for detection and prediction of their CNS relapse. Our findings indicate that the evaluation of oncogenic microRNAs in CSF and plasma potentially provides a sensitive tool for the early detection of secondary CNS lymphoma, the monitoring and estimating of treatment efficacy, and the prediction and early detection of CNS relapse.Lymphoma with secondary central nervous system (CNS) involvement represents one of the most aggressive malignancies, with poor prognosis and high mortality. New diagnostic tools for its early detection, response evaluation, and CNS relapse prediction are needed. We analyzed circulating microRNAs in the cerebrospinal fluid (CSF) and plasma of 162 patients with aggressive B-cell non-Hodgkin’s lymphomas (B-NHL) and compared their levels in CNS-involving lymphomas versus in systemic lymphomas, at diagnosis and during treatment and CNS relapse. We identified a set of five oncogenic microRNAs  in CSF that detect, with high sensitivity, secondary CNS lymphoma involvement in aggressive B-NHL, including DLBCL, MCL, and Burkitt lymphoma. Their combination into an oncomiR index enables the separation of CNS lymphomas from systemic lymphomas or nonmalignant controls with high sensitivity and specificity, and high Receiver Operating Characteristics . Longitudinal analysis showed that oncomiR levels reflect treatment efficacy and clinical outcomes, allowing their monitoring and prediction. In contrast to conventional methods, CSF oncomiRs enable detection of early and residual CNS involvement, as well as parenchymal involvement. These circulating oncomiRs increase 1–4 months before CNS relapse, allowing its early detection and improving the prediction of CNS relapse risk in DLBCL. Similar effects were detectable, to a lesser extent, in plasma. Central nervous system (CNS) involvement presents a diagnostic and therapeutic challenge in regard to both primary (PCNSL) and secondary CNS lymphomas (SCNSL). Their prognosis is poor and median overall survival (OS) is 4–5 months; 2-year OS is 10–20%, with a less favorable prognosis for SCNSL  stands for the levels of any of the oncomiRs featuring in the respective index. If the coefficient for one of the oncomiRs was determined to be 0, the respective oncomiR was excluded from the index calculation. MiR-let-7a-normalized and Ctrl/average systemic-equalized data were employed. The indices with oncomiRs combinations with the highest CNS vs. systemic separation (ROC) were finally selected. Further details of coefficient determinations are provided in the The following oncomiR indices formulae were used see :CSF:Plasma:To identify circulating microRNAs that could be used to detect secondary CNS lymphoma involvement, we analyzed lymphoma-associated microRNAs in cerebrospinal fluid (CSF) in aggressive B-NHL subtypes  with and without CNS involvement and compared their levels at diagnosis, during treatment, and during CNS relapse.Five microRNAs  were selected from 20 candidate microRNAs analyzed in the initial screen see the  accordinn = 20, DLBCL = 15, MCL = 4, B-NHL-NOS = 1).The patient cohort included 162 B-NHL patients consisting of 108 systemic and 54 secondary CNS lymphoma (SCNSL): DLBCL = 97 , MCL = 34 , BL = 18 , B-NHL-NOS = 13 . The SCNSL cohorts included patients with CNS involvement at the time of initial diagnosis of systemic lymphoma (SCNSL-dg) and patients with newly detected CNS relapse (n = 22). All SCNSL subtypes had significantly increased microRNAs of all five varieties  in CSF  extent as in DLBCL compared to systemic lymphoma , despiteIn order to evaluate the abundance of individual oncomiRs as a single variable, we developed a logistic regression model that combines individual microRNA abundances into a single classifier, which we called the oncomiR index (indices). It evaluates the prognostic value of individual microRNAs for particular B-NHL subtypes, to maximize the difference between CNS-positive and systemic lymphomas .The oncomiR index overall significantly improved the separation of CNS lymphoma vs. systemic lymphoma in all lymphoma subtypes  and provided high sensitivity/specificity   and patients with newly detected CNS relapse . In addition, we stratified CNS lymphoma with regard to parenchymal and lepto-meningeal involvement. Both lymphoma localizations displayed a comparable significant increase of oncomiR indices C, as welIn summary, the analysis of microRNA in CSF revealed that five oncogenic microRNAs combined into the oncomiR index can accurately detect secondary CNS lymphoma involvement in all analyzed B-NHL subtypes.Since collection of CSF is an invasive method, the possibility of testing of oncomiRs in peripheral blood (PB) would be beneficial. Therefore, we investigated the abundance of oncomiRs in plasma (paired samples to CSF). We were able to detect similar trends in the increase of all plasma oncomiRs in all CNS lymphoma subtypes, compared to controls as in CSF .The oncomiR increase and separation of CNS from systemic lymphoma in plasma was lower than in CSF. Although an increase in oncomiR levels was observed in all cases except for BL, it was less significant .OncomiR indices in plasma reflected individual oncomiRs’ performance. There was a significant increase in oncomiR indices in all CNS lymphoma compared the control group A and a hp value, except for the BL oncomiR index that performed superior CNS vs. systemic separation and sensitivity/specificity  compared to controls were similarly high in plasma as in CSF, they were lower compared to systemic lymphoma, with the exception of BL-SCNSL and CNS relapses , indicating that plasma can be used for the detection of CNS lymphoma involvement only in these cases.We next tested the dynamics of oncomiRs in CSF and plasma during therapy. The CSF and plasma samples were collected at multiple time-points: at the diagnosis, during the administration of intrathecal therapy, or during follow-up examinations.therapy-responders (n = 8), the clinical manifestation of remission was preceded by a gradual decrease in oncomiRs. While FCM and cytology usually switched rapidly from positive to negative findings, the oncomiR levels gradually decreased from diagnosis to remission.Interestingly, in An example of such patients is shown in A similar trend was also observed in plasma. Interestingly, an oncomiR decrease in plasma was delayed by several days compared to CSF, and the clearance of plasma microRNAs was preceded by their initial increase (before treatment began to work), indicating different response dynamics in the CNS and the system. Decreasing oncomiR levels in CSF and plasma predicted complete remission ten months in advance.refractory lymphoma (n = 5) displayed a gradual increase of oncomiRs in CSF during treatment, as documented by the sharp increase of oncomiRs in the Burkitt lymphoma patient  with secondary CNS involvement   B. Despitlvement) . NotablyCNS relapse. n = 4) that relapsed/progressed to CNS. This patient  achieved a partial remission after first line treatment. Three months later, CNS and systemic progression were confirmed . The oncomiR levels in plasma initially decreased in response to therapy, while their subsequent increase preceded systemic and CNS progression by 1 month. The high plasma levels of oncomiRs at diagnosis could predict later progression. Despite transient improvement, the patient died 1 month after the last sampling due to the progression of systemic lymphoma. In contrast to plasma, where oncomiRs reflected systemic remission, in CSF the oncomiR levels already increased before and at the time of systemic remission. Thus, the oncomiR increase preceded the lymphoma CNS progression (detected by cytology and MRI) by 3 months. OncomiRs in CSF subsequently decreased with the reduction of CNS involvement. A similar increase in circulating oncomiRs prior to CNS relapse was also observed in the remaining patients.The predictive potential of oncomiRs in CSF and plasma is further visible in systemic patients who developed A total of seven patients with serial samples before CNS relapse (four systemic to CNS relapses and three SCNSL relapses) were detected. In all cases, the gradual oncomiR increase was observed before CNS relapse, in CSF with a median of 3 (range 1–4) months and in plasma 2.7 (1–4) months prior to CNS relapse.A comparison of the cumulative levels of tested oncomiRs at different stages of the clinical course D shows tThe longitudinal analysis indicates that: 1. OncomiR levels can change rapidly in response to the disease’s state and therapy; 2. Trends in oncomiR levels reflect and predict therapy efficacy and patient outcomes evaluated by conventional methods; 3. CNS relapse or CNS progression of secondary CNS and systemic lymphoma is preceded by 1–4 months of increasing oncomiR levels in both CSF and plasma.n = 72) and DLBCL with secondary CNS involvement  at the time of diagnosis. We compared the subgroup that did not develop CNS relapse with the subgroup that subsequently relapsed to CNS , which were termed subsequent relapses.We hypothesized that in addition to elevated oncomiRs shortly before and during CNS relapse, there is already a predisposition to CNS relapse due to increased levels of CNS-specific oncomiRs at the time of lymphoma diagnosis. To test this hypothesis, we analyzed oncomiR levels in CSF and plasma of systemic DLBCL (The subsequent CNS relapses of secondary CNS DLBCL (DLBCL-SCNSL) had oncomiR levels among the highest compared to the non-relapsing group, both in CSF and plasma, with a significant increase in plasma miR-19a and miR-92a . In casep = 0.012, miR-21) but not in CSF . Therefore, we performed an univariate analysis (Kaplan-Meier) of oncomiRs in plasma. Patients were stratified according to the oncomiR level (high vs. low) at diagnosis (p = 0.009) versus 6.7% in the low oncomiR group  indicated the significant effect of the oncomiRs on the time to CNS relapse in plasma  with a high 4-year relapse rate of 51.5%  . The relapse rates were higher when compared to the time of diagnosis: CSF 60.6% (HR 31.3) and plasma 55.2%, (HR 16.5) for the high microRNA group (vs. 0% for low microRNA for both CSF and plasma) . This sup = 0.0001, Plasma HR = 2.5; p = 0.0014) than patients with low oncomiRs  in DLBCL. The microRNA high/low threshold was prognostic for OS, and patients with high oncomiRs had lower survival  and OS (CSF + plasma) and the combination of plasma oncomiRs with CNS-IPI significantly improves the prediction of CNS relapse risk.We focused on finding a sensitive marker of lymphoma CNS involvement in secondary CNS B-NHLs and their CNS relapses. We provide evidence that the expression of five oncogenic circulating microRNAs  is elevated to a similar extent in CSF of aggressive B-NHLs with secondary CNS involvement including DLBCL, MCL, and BL. These microRNAs are early and sensitive markers of CNS lymphoma involvement and allow the separation of CNS-involving lymphomas from systemic-only lymphomas. The separation from control samples  was even higher, which is consistent with earlier reports from PCNSL ,23.To assess the abundance of tested microRNAs cumulatively, and to account for the variable prognostic power of individual microRNAs in particular lymphoma types, we employed a logistic regression model that combined individual microRNA into a classifier termed the oncomiR index. This approach provided a high separation of the SCNSL vs. systemic lymphoma involvement . The high level of CNS/systemic separation allowed the threshold to be set to favor either high sensitivity, specificity, or a balance of the two variables to obtain the optimal diagnostic yield. For example, in the case of DLBCL CSF, the used threshold of 8.2 provided balanced sensitivity (91.3%) and specificity , while the threshold 6.9 provided 100%/82% (sensitivity/specificity).These findings indicate that these circulating CSF oncomiRs, incorporated into the oncomiR index, can serve as an indicator of CNS involvement of aggressive B-NHL , and potentially other types of lymphoma, as supported by similar trends that we observed in follicular lymphoma (not shown). These results are consistent with previous reports showing that two of the five microRNAs tested in this study (miR-21 and miR-92a) are elevated in the CSF of PCNSL ,23.Although not tested, we suggest that the combination of several oncomiRs may provide specificity to distinguish CNS lymphoma from other diseases involving the CNS. For example, the most common glioblastoma has elevated CSF miR-21  but not Although the studied B-NHLs represent distinct lymphoma subtypes, they are all of B-cell origin and, despite differences, they share common features. All tested oncomiRs have been reported to be upregulated in biopsies or blood of studied systemic lymphoma subtypes. MiR-17-92 was the first described to be upregulated in various lymphoma subtypes ,29, withThe different oncomiRs included in the oncomiR indices or their varying strengths (represented by coefficients) between different lymphoma subtypes probably reflect molecular differences in the physiology of individual lymphoma subtypes. The differences between CSF and plasma index coefficients likely reflect the differences in the complexities of CSF and plasma, which may reflect the sum of circulating microRNAs from different sources contained in CSF and plasma . The determination of oncomiR index coefficients also depends on the number of patients studied, and therefore validation of the study in a larger number of patients may lead to more accurate oncomiR indices (coefficients) and the thresholds for lymphoma CNS involvement.Using ROC analysis, we determined the threshold values of the oncomiR index for detecting CNS lymphoma involvement. These thresholds are valid for both concurrent systemic and secondary CNS involvement at the time of initial diagnosis of SCNSL, as well as for newly detected CNS relapses. We note that the index thresholds apply only to oncomiR index values, but not to individual microRNAs, and due to the different microRNAs incorporated into the indices or their different coefficients, the thresholds vary between lymphoma subtypes.Conventional diagnostic methods for CNS lymphoma involvement have limitations. The most commonly used methods, flow cytometry and cytology of CSF, are not able to detect the intra-parenchymal involvement that is detectable only by magnetic resonance (with low specificity/sensitivity) followed by brain biopsy ,6. MicroDue to the invasiveness of lumbar puncture, the testing of lymphoma CNS involvement by microRNAs from blood would be beneficial. Indeed, we were able to detect increased levels of tested microRNAs in both plasma and serum (data not shown). The relative microRNA abundance in plasma and serum was comparable. We preferred to use plasma samples because there was a higher variability in microRNA levels in the serum . We speculate that this is probably due to the fact that extracellular microRNAs are incorporated in protein  complexes ,42 whichAlthough the oncomiR increase and separation of CNS lymphoma from control samples was, in plasma, comparable to that in CSF, the separation of CNS from systemic lymphoma in DLBCL, MCL, and B-NHL-NOS was lower in plasma. This finding is possibly caused by several reasons: 1. systemic lymphomas have higher levels of oncomiRs in plasma than in CSF ; 2. The heterogeneity of lymphoma cells. Our data indicate that the CNS is likely invaded by lymphoma cells/clones with a higher expression of oncomiRs (likely due to their higher aggressiveness); 3. Plasma potentially contains circulating microRNAs produced by other organs . Thus, plasma microRNA levels are complex and reflect the sum of the microRNAs produced by systemic lymphoma, other organs, and potentially the microRNA from CNS lymphoma (if the blood brain barrier is compromised or permissive for microRNA), due to the higher volume of plasma diluted. As a result, we observed a high diagnostic CNS vs. systemic separation in plasma in BL (oncomiR index AUC = 1.0), and acceptable in MCL (AUC = 0.84) but not DLBCL (AUC = 0.78) and B-NHL-NOS (AUC = 0.67).Longitudal microRNA analysis during the treatment of CNS lymphoma revealed the following phenomena: oncomiR levels in CSF reflect, with high accuracy, the disease state and response to treatment, thus allowing their monitoring. Furthermore, the trends in microRNA expression preceded and thus predicted subsequent remission or progression in advance. In particular, microRNAs gradually decreased in the case of responsive CNS lymphomas and, reciprocally, gradually increased in the case of refractory CNS lymphomas. OncomiRs were able to monitor treatment efficacy at a higher resolution  and over a longer time window than FCM or cytology; e.g., in advance or after (on average up to 3 months) positive FCM/cytology findings, detecting likely initial or residual disease. In numerous samples, microRNAs were able to detect CNS involvement that was not detected by FCM or cytology of CSF. For example, oncomiRs predicted treatment failure up to 3 months earlier than FCM or cytology B.Both CSF- and plasma-derived oncomiRs have the ability to monitor the response to treatment A–D. HoweThe sensitivity and timeliness of microRNA evaluation in CSF (and plasma) is further documented by increased oncomiR levels in advance of CNS relapse. First, CNS relapses  have significantly increased oncomiRs in CSF and plasma at the time of relapse detection. In addition, long-term analysis showed that oncomiR levels begin to increase 1–4 months prior to the clinical demonstration of the relapse in both CSF and plasma (at times when FCM and cytology were negative), suggesting that increasing oncomiR levels indicate emerging CNS relapse. Thus, circulating oncomiRs enabled the detection of CNS relapse not only at the time of clinical relapse, but also several months in advance, allowing a potentially earlier application of therapy. We speculate that this predictive potential is likely due to the sensitivity of microRNA analysis to detect occult initial stages of CNS involvement, not detectable by conventional methods. It should be noted that while multiple oncomiRs were increased at the time of detection of secondary CNS involvement, this effect decreased with prolonged time before CNS relapse. At the time of diagnosis of systemic DLBCL, several months to several years before CNS relapse, only plasma miR-21 and CSF miR-155 were significantly elevated.We further explored the ability of oncomiRs to predict CNS relapse at the time of diagnosis of systemic DLBCL. The risk model, based on stratification of patients by high/low oncomiR in plasma, indicated that circulating oncomiRs (as an independent risk factor) could provide a comparable estimate of relapse rate as CNS-IPI (31.8 vs. 28.1%). Interestingly, the oncomiR model identified a lower number of non-relapsing patients in the high-risk group than CNS-IPI (23.2% vs. 37.5%). The predictive oncomiR potential increased with the time to approaching relapse , and oncomiR levels at diagnosis were lower than at the time of CNS relapse. Unlike plasma, the CSF oncomiRs at diagnosis were not significant for CNS relapse prediction. This is consistent with a scenario in which CNS is not initially affected and lymphomas invade the CNS from systemic disease, which is likely predisposed to CNS relapse by higher oncomiRs, and CSF oncomiRs subsequently increase at the time when lymphoma spreads to the CNS.In addition, we combined CNS-IPI and plasma oncomiR stratification of relapse risk to one prediction model. Notably, the incorporation of oncomiRs into the CNS-IPI/oncomiR model significantly improved the 4-year relapse risk prediction (nearly two-fold), compared to the single CNS-IPI or oncomiR models (51.5% vs. 28.1% and 31.8% respectively). It substantially reduced the high-risk group to only 17% of all patients (compared to 42.9% in CNS-IPI and 28.6% in the oncomiR model), by reducing the non-relapsing patients in the high-risk group. This may help to limit the use of CNS-oriented prophylactic treatment to only those high-risk patients who need it and avoid the over-treatment of patients who cannot benefit from it. A similar approach integrating biomarkers into CNS-IPI was recently described ,13 to beFinally, we found that high levels of oncomiRs are prognostic for overall survival (OS) in both CSF and plasma of DLBCL, further confirming the prognostic and CNS lymphoma detection capabilities of circulating oncomiRs.A summary of the possible use of CSF and plasma oncomiRs as markers of CNS lymphoma involvement is provide in the This study describes a set of circulating oncogenic microRNAs in CSF and plasma that (combined into the oncomiR index) can serve as sensitive markers of secondary CNS B-NHL, including DLBCL, MCL, and BL. In contrast to conventional diagnostic methods, these oncomiRs are able to detect early and residual CNS lymphoma involvement, as well as parenchymal involvement in DLBCL. Levels of these oncomiRs reflect and predict clinical responses to therapy. In addition, oncomiRs are increased before CNS relapse and in DLBCL (plasma) oncomiRs improve in combination with CNS-IPI the prediction of CNS relapse risk.In summary, the study indicates a potential use of microRNA evaluation in CSF and plasma for early detection of secondary CNS involvement in aggressive B-NHL lymphomas, as well as for the monitoring and predicting of therapy efficacy and for the prediction of CNS relapse or its early detection.


Acute-on-chronic liver failure (ACLF) has a high mortality rate. The role of granulocyte colony-stimulating factor (G-CSF) in ACLF remains controversial. Monocytes/macrophages are core immune cells, which are involved in the initiation and progression of liver failure; however, the effect of G-CSF on monocytes/macrophages is unclear. The study aimed to verify the clinical efficacy of G-CSF and explore the effect of it on monocytes in hepatitis B virus (HBV)-related ACLF (HBV-ACLF) paitents.in vivo and in vitro experiments.We performed a large randomized controlled clinical trial for the treatment of HBV-ACLF using G-CSF. A total of 111 patients with HBV-ACLF were prospectively randomized into the G-CSF group  or the control group (standard therapy). All participants were followed up for at least 180 days. The relationship between monocyte count and mortality risk was analyzed. The effect of G-CSF on the phenotype and function of monocytes from patients with HBV-ACLF was evaluated using flow cytometry P = 0.0142). In the G-CSF-treated group, the monocyte counts on days 0 and 7 were independently associated with an evaluated mortality risk in the fully adjusted model (Model 3) . Further analysis showed that after treatment with G-CSF in HBV-ACLF patients, the expression of M1-like markers (HLA-DR and CD86) in monocytes decreased . The expression of MerTK (M2-like marker) increased (P = 0.0002). The secretion of TNF-α, IL-6, and IL-10 from monocytes decreased without lipopolysaccharide (LPS) stimulation  or with LPS stimulation . Similar effects were observed in vitro experiments.The survival probability of the G-CSF group at 180 days was higher than that of the control group  transition of monocytes, which may contribute to the recovery of ACLF.ClinicalTrials.gov, identifier (NCT02331745). Acute-on-chronic liver failure (ACLF) is a clinical syndrome characterized by severe hepatic dysfunction resulting from acute injury to an underlying chronic liver disease and a substantially high short-term mortality rate , 2. The Although traditionally considered a hematopoietic regulator, granulocyte colony stimulating factor (G-CSF) has been regarded as a candidate treatment for ACLF recently. Several studies have established the efficacy and safety of G-CSF in the management of ACLF –9. Howev++CD16− , CD14++CD16+ (intermediate), and CD14+CD16++   , 20. Monassical) . TherefoIn Asia, the main etiology of ACLF is chronic hepatitis B virus (HBV) infection –23. The The current study includes a clinical trial and basic experiments. The former was a randomized, controlled, open-label trial to evaluate the efficacy of G-CSF for HBV-ACLF. Then, we evaluated the effect of G-CSF on the phenotype and function of monocytes from patients with HBV-ACLF.In the clinical trial, all participants were recruited from the Fifth Medical Center of the People’s Liberation Army (PLA) General Hospital for screening from June 2014 to September 2016. Eligible patients met the diagnostic criteria for ACLF, suggested by the Asian Pacific Association for the Study of the Liver (APASL) , which aAscites was identified by clinical manifestations, diagnostic paracentesis, and abdominal imaging examination; Acute kidney injury was diagnosed according to the International Club of Ascites (ICA) corresponding standard ; A West 3. (ii) pneumonia: pulmonary imaging changes  plus at least 1 of the following presentation  plus at least 1 of the following symptoms or signs  and/or etiological evidence. (iii) urinary tract infection: Patient had at least 1of the following clinical presentations  and positive urine culture; or at least 2 of the above presentations and more than 10 leukocytes/μL in urine. (iv)bacteremia: positive blood culture  spontaneous bacterial peritonitis: polymorphonuclear cell count in ascitic fluid ≥250/mm culture , 30.PASS 11.0 software  was used to calculate the sample size. Based on our previous study, we expected that G-CSF therapy could improve the survival rate at 180 days by approximately 20% in the G-CSF group compared to that in the control group. With a statistical power of 80%, we required 52 patients in each group to detect this meaningful difference at a significance level of 5%. Considering the possible loss of patients to follow-up (10%), 114 patients were included in this study. The computer-generated randomization number code was prepared for each patient. The patients were randomly allocated to the G-CSF group  or control group .G-CSF  was injected subcutaneously at a dose of 5 μg/kg every day for 6 days and then every other day until day 18  in G-CSF group patients; thereafter, physical examination and laboratory tests were conducted. All patients received standard medical therapy, including intensive care monitoring, antiviral therapy, supportive therapy, and prevention and treatment for complications. All patients with infection were treated with antibiotics. Patients received albumin, terlipressin, and so on if required.A total of 114 patients meeting the inclusion and exclusion criteria were enrolled in the study  were obtained from patients with HBV-ACLF (n = 12), before and after G-CSF treatment, to evaluate the effect of G-CSF on monocytes. Surface markers and intracellular cytokine expression in monocytes, expressed as frequency or mean fluorescence intensity (MFI), were measured using flow cytometry on the BD FACSymphony A5 analyzer . Data were analyzed using the FlowJo software .For surface marker staining, APC-Cy7-conjugated anti-CD45 (BD Biosciences), PerCP-Cy5.5-conjugated anti-CD14 (BD Biosciences), APC-conjugated anti-CD16 (BD Biosciences), BV711-conjugated anti-HLA-DR (BD Biosciences), PE-conjugated anti-MerTK (R&D Systems), PE-Cy7-conjugated anti-CD163 (eBioscience), BV421-conjugated anti-CCR2 (BioLegend), FITC-conjugated anti-CX3CR1 (BioLegend), and BUV-737-conjugated anti-CD86 (BD Biosciences) antibodies were used. For intracellular cytokine staining (ICCS), BV-421-conjugated anti-IL-6 (BD Biosciences), PE-CF594-conjugated anti-IL-10 (BD Biosciences), and BV-510-conjugated anti-TNF-α (BioLegend) were used. Dead cells were excluded using Fixable Viability Stain 440UV (BD Bioscience).For surface marker labeling, PBMCs were incubated with surface fluorescent-labeled monoclonal antibodies. For intracellular staining, PBMCs were incubated with or without 100 ng/mL lipopolysaccharide (LPS) and protein transport inhibitor  for 6 h, followed by staining with surface markers, and fixation, permeabilization, and staining with the corresponding fluorescent intracellular antibodies.+ monocytes were further isolated using magnetic bead separation . The purity of monocyte separation was examined using flow cytometry, and CD14+ monocytes with a purity >95% were used in vitro experiments. Isolated monocytes were incubated in complete media with 20 ng/mL G-CSF or an equivalent volume of PBS (control) for 24 h for surface staining. For intracellular staining, the isolated CD14+ monocytes were stimulated with G-CSF (20 ng/ml) for 18 h. After 18 h incubation, LPS (100 ng/ml) and protein transport inhibitor  was added and continue to incubation for 6 h, followed by staining with surface markers, and fixation, permeabilization, and staining with the corresponding fluorescent intracellular antibodies. After all the above steps, the monocytes were harvested and analyzed for their surface phenotype and intracellular cytokine levels using flow cytometry. Staining was performed as previously described.PBMCs were obtained from patients with HBV-ACLF who did not receive G-CSF therapy. CD14The effect of G-CSF therapy on monocyte phagocytosis and the oxidative burst capacity of patients with HBV-ACLF was evaluated. Monocyte phagocytosis and oxidative burst capacity were tested using the PHAGOTEST kit  and PHAGOBURST kit  following the manufacturer’s protocol, respectively, and then assessed by flow cytometry.ClinicalTrials.gov (NCT02331745).The study protocol and informed consent form were approved by the Human Ethics Committee of the Fifth Medical Center of the PLA General Hospital. All the procedures were in accordance with the ethical guidelines of Declaration of Helsinki. Written informed consent was obtained from patients or their guardians before enrollment. This trial was registered at t-test  or Mann–Whitney U test  were used to compare differences between the two groups. Patients lost to follow-up contributed to the censored data. Survival rates were calculated using the Kaplan–Meier method and compared using the Log-Rank test. In the crude model and three multivariate adjusted models, Cox proportional hazards model was used for multivariate regression analysis to estimate the hazard ratios  and 95% confidence intervals  for the risk of 180-day mortality. Statistical analyses and graphing were conducted using the statistical packages R version 3.4.3 , EmpowerStats  and the MedCalc 15.2.2 . Statistical significance was set at P < 0.05.In the clinical trial, continuous variables with normal distribution and skewed distribution were expressed as mean ± standard deviation (SD) and median [interquartile range (IQR)], respectively. Categorical variables were expressed as numbers and percentages. The chi-square test  and Student’s U tests, were applied to comparisons across the different groups. Comparisons within the same individual were performed using Wilcoxon’s matched-pair test. Statistical significance was set at P < 0.05.In the Experimental section, statistical analysis was performed using GraphPad Prism 7.00 . Data are presented as median (IQR). Non-parametric analyses, such as Kruskal–Wallis and Mann–Whitney P = 0.066 and 0.051, respectively). Meanwhile, the serum creatinine level in the control group was slightly higher than that in the G-CSF group, but both were within the normal range, and no statistical difference was detected (P = 0.090). As shown in The patients tolerated the treatment well, and no severe side effects were observed. One of the patients developed a mild rash during the follow-up and discontinued all drugs, including G-CSF. However, no evidence was shown that the rash was induced by G-CSF. The results showed that the G-CSF treatment protocol was safe.P > 0.05) between the control and G-CSF groups.During the first month, the main new-onset complications were infection, hepatic encephalopathy, and acute kidney injury , P = 0.0002 in Model 3]. However, after six consecutive days of treatment, the control group did not demonstrate significant changes, whereas the positive association between monocyte count on day 7, and the risk of death was significantly weakened in the G-CSF-treated group . Similar relationship was observed between monocyte count and 90-day mortality , the intermediate and non-classical monocytes demonstrated a decreasing trend, whereas classical monocytes demonstrated an increasing trend. Thus, a shift in monocyte subpopulations was detected after G-CSF treatment.According to traditional gating, monocyte subtypes can be identified by the expression of CD14 and CD16 , whereas the expression of M2-like marker (MerTK) increased . There was no significant change in the expression of CCR2 and CX3CR1 in monocytes according to MFI before and after G-CSF treatment . The above results indicate that circulating monocytes after G-CSF therapy showed an anti-inflammatory/pro-restorative phenotype in patients with HBV-ACLF.To fully evaluate the effect of G-CSF on monocytes in HBV-ACLF, we designed detailed immunophenotypic analyses to detect the levels of activation/inflammation markers  and tissue-homing markers (CCR2 and CX3CR1) before (day 0) and after the administration of G-CSF (n = 12). As shown in P < 0.0001; IL-6: day 0 vs. day 3 vs. day 7 vs. day 14 vs. day 28 = 24.0% vs. 7.5% vs. 10.9% vs. 8.4% vs. 44.6%, respectively; P = 0.0025; IL-10: day 0 vs. day 3 vs. day 7 vs. day 14 vs. day 28 = 0.9% vs. 0.5% vs. 0.2% vs. 0.5% vs. 1.6%, respectively; P = 0.0004). In contrast to pre-treatment (day 0), cytokine secretion in monocytes showed a decreased response to LPS stimulation after G-CSF treatment . Hence, G-CSF reduces the inflammatory response of monocytes in HBV-ACLF patients.We evaluated whether G-CSF treatment in patients with HBV-ACLF modulated the ability of monocytes to produce IL-6, TNF-α, and IL-10 following LPS challenge or not. As shown in in vitro experiments to further clarify the effect of G-CSF on the phenotype and function of monocytes in patients with HBV-ACLF. As shown in P < 0.01). There was no significant difference in the expression of homing receptors CCR2 and CX3CR1 between the two groups .We tested and compared phagocytosis  phenotype, which may contribute to the attenuation of liver injury and promote recovery of ACLF. These novel findings expand our knowledge of the role of G-CSF in the treatment of liver failure and provide new evidence that G-CSF may contribute to protection against liver damage.P = 0.001). Another study also revealed superior 90-day survival (P = 0.04). In addition, these two studies showed improvement in liver function, as indicated by the MELD and Child-Turcotte-Pugh (CTP) scores. However, a European study by Engelmann et al. (P = 0.805). The liver function scores also did not improve in the G-CSF treatment group. The positive effect of G-CSF has only been confirmed in Asian studies, and not in the European study.In the current study, we observed a survival benefit in patients with HBV-ACLF after G-CSF therapy. This result was in agreement with those of several previous studies , 6. A stsurvival  of HBV-An et al.  failed t+ cells markedly increased in the liver tissue after G-CSF therapy in patients with ACLF. Subsequently, they demonstrated that G-CSF enhanced the mobilization of bone marrow hematopoietic stem cells and promoted their homing in the hepatic parenchyma. G-CSF therapy was also shown to enhance dendritic cell recruitment to the liver and suppress interferon-γ secretion by CD8+ T cells to attenuate hepatocellular injury  in monocytes was markedly suppressed  in monocytes in animal models of ischemic stroke  production  transition of monocytes, which may contribute to the recovery of ACLF. However, owing to heterogeneity, monocytes play distinct roles in ACLF, from pro-inflammatory to pro-resolution, depending on the microenvironment. Therefore, a full understanding of the pathophysiological processes and developmental stages of ACLF may help to select the right patients for appropriate therapy.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by the Human Ethics Committee of the Fifth Medical Center of the PLA General Hospital. The patients/participants provided their written informed consent to participate in this study.JT, HW, XX, JC (6th author), XM, ZL, HS, XL and CL participated in the data acquisition. JH, ZW, HF and JT designed the study. JT, HW and HF performed analyses and interpretation of data. JT, HW, XM, JC (9th author) and HXW completed the experiments. JT, HW, ZW wrote the first draft of the manuscript and incorporated revisions. JT, HW and JH prepared the final version. All authors contributed to the article and approved the submitted version.This work was supported by a grant from the Capital’s Funds for Health Improvement and Research, China (NO.2020-1-5031) and National Clinical Research Center for Infectious Diseases (NCRC-ID202106).The 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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


The association of polymorphisms in molecules involved in the immune response (checkpoint inhibitors) with the clinical outcome after allogeneic transplantation  has been described. Lymphocyte Activation 3 (LAG3) is a surface protein that plays a regulatory role in immunity as an inhibitory immune checkpoint molecule.To determine its role in the alloHSCT setting, we analyzed 797 patients transplanted from HLA-identical sibling donors. The LAG3 rs870849 C>T polymorphism was genotyped in donors.We detected a higher incidence of severe acute GVHD in patients transplanted from donors with TT genotype . Overall survival (OS) was worse for patients transplanted from donors with the rs870849 CT/TT genotype , as well as disease-free survival (DFS)  and transplant-related mortality (TRM) . When combining the LAG3 rs870849 and the PDCD1 rs36084323 genotypes of the donor, three genetic groups were well defined, allowing a good stratification of the risk of acute GVHD, TRM, OS and DFS.We conclude that the LAG3 genotype of the donor may be considered in donors’ selection. As this selection may be limited in the HLA-identical sibling donor scenario, further studies exploring the impact of LAG3 genotype of the donor in unrelated transplantation are warranted. CTLA4) gene, and the programmed cell death 1 (PD-1) gene of the donor have been associated with differences in the clinical outcome after allogeneic hematopoietic stem cell transplantation   –5.Lymphocyte Activation 3 (LAG-3), also known as CD223, belongs to the immunoglobulin superfamily and contains 4 extracellular immunoglobulin domains and is expressed in T lymphocytes, B lymphocytes, NK cells, and plasmacytoid dendritic cells. The LAG3 gene is located next to CD4 in chromosome 12 and contains 8 exons . LAG3 trStructurally, LAG-3 is a CD4-homologous protein, although it shares only 20% of the aminoacidic sequence. Following lymphocyte activation, LAG3 interacts with molecules of the major histocompatibility complex of class II (MHC-II) by a few residues of the D1 domain, in the most distal part of the cell membrane of the lymphocyte, generating an inhibitory signal through its intracellular portion and therefore, bringing the lymphocyte to a state of anergy –12. In aLAG3 is stored in the trans-Golgi vesicles (late endosome) of CD8+ lymphocytes together with PD-1, and the action of the two molecules is synergistic by inhibiting lymphocyte activation , 16.Like CTLA-4, LAG3 has two main isoforms. The transmembrane form, of 70 kDa, is formed by the 4 immunoglobulin domains (D1-D4) associated with the intracytoplasmic portion (KIELLE) which will deliver the inhibitory signal. The soluble form of LAG3 (sLAG3) is derived from the proteolytic cleavage of surface LAG3 mediated by disintegrin and metalloproteinase ADAM10 and ADAM17 at the connecting peptide between D4 and the transmembrane domain .LAG-3 has emerged as an important regulatory molecule of the immune response, comparable to CTLA-4 or PD-1, consisting in the inhibition of T-cell activation. As it has been described for PD-1, constitutive expression of LAG-3 is associated with exhausted T cells and Treg. The LAG-3 gene has 8 exons, and several single-nucleotide polymorphisms (SNPs) have been described. The C/T transition encoded by the SNP rs870849 encodes a substitution of the amino acid isoleucine for the amino acid threonine in the transmembrane domain of the LAG-3 protein (Thr455Ile). Zhang et al. described an association between the LAG-3 rs870849 TT genotype and an increased risk of multiple sclerosis , but theTo date, there are not data exploring whether LAG-3 may be a molecule involved in the development of GVHD in patients receiving an allogeneic stem cell transplant. This study aims to investigate whether donor’s LAG-3 genotype influences clinical outcome after alloHSCT from HLA-identical sibling donors.LAG3 genotype.We retrospectively studied a cohort of 797 patients receiving an allogeneic HSCT from an HLA-identical sibling donor between 2000 and 2014 in Spanish centers. The conditioning regimen was myeloablative in 70.1% of cases and peripheral blood was the most used stem cells source. The clinical characteristics of the studied cohort are summarized in Written informed consent was obtained from patients and donors before DNA storage. DNA samples were obtained from peripheral blood using the QIAamp DNA Blood Mini Kit , in accordance with the manufacturer’s instructions, and stored at -80°C until use. Samples from patients included in this study were provided by the IDIBGI Biobank , integrated in the Spanish National Biobanks Network and they were processed following standard operating procedures with the appropriate approval of the Ethics and Scientific Committees. The study was approved by the Ethics Committee of the Dr. Josep Trueta University Hospital  and fulfilled the recommendations of the Helsinki declaration.According to the allele frequency and previous association with autoimmune diseases, we decided to explore the rs870849 C>T polymorphism, located in the transmembrane domain.via allelic discrimination plots on the QuantStudioTM 6 and 7 Flex Real-Time PCR System  by using TaqMan real-time PCR primers and probes obtained as commercially available (TaqMan SNPmGenotypingAssay (rs870849); reference number: 4351379 . The Assay reagents included both the necessary primers and fluorescently labeled (FAM and VIC) TaqMan MGB probes to amplify and detect each polymorphism. The PCR cycling conditions were as follows: initial denaturation at 95°C for 10 min, followed by 40 cycles of denaturation at 95°C for 15 s and annealing/extension at 60°C for 60 s. The presence of wild-type and variant alleles was defined by comparing the relative end-point fluorescence created by the degradation of each fluorescently labeled TaqMan probe, according to the manufacturer’s instructions.The genotype of the donors for this polymorphism was determined Allele and genotypes frequencies were formulated by direct counting.Homogeneity between genotype groups was performed using the Chi-square test for qualitative variables and Student’s T-test or ANOVA for continuous variables.Univariate analyses were performed to evaluate the association between the rs870849 genotype of the donor and acute GVHD (aGVHD), overall survival (OS), disease-free survival (DFS), Transplant-related mortality (TRM) and relapse.The Kaplan-Meier method was applied for the analysis of OS and DFS. Curves were compared using the log-rank test. Cumulative incidence estimates were used to explore differences in aGVHD, TRM and relapse. Death without signs of aGVHD was considered as a competitive risk for acute GVHD. The competing risk for TRM was death after relapse and the competing risk for relapse was death in complete remission. Patients with non-malignant diseases were excluded in the relapse analysis. Chronic GVHD incidence was considered as a qualitative variable instead a time-dependent one because the onset of this complication was often missing or not clear, comparing incidence between genetic groups by the chi squared test in univariate analysis and logistic regression model in multivariate analysis.Multivariate analysis with regression modeling of competing risks  was perfLAG3 rs870849 genotype could be successfully determined in all the analyzed donors. As previously described in Caucasian population, the C allele was the majoritarian, being detected in 83.5% of cases whereas the T allele was identified in 63.7% of the studied cases. The genotype distribution showed 290 donors (36.4%) homozygous for the C allele, 132 homozygous for the T allele (16.6%) and 375 heterozygous CT (47.1%).LAG3 rs870849 genotype of the donor except a higher proportion of patients with the combination Male recipient - Female donor in the group with donors homozygous for the T allele , heterozygous CT (32.5%) or homozygous for the T allele (33.8%) .All patients showed successful engraftment. The cumulative incidence of grades II, III or IV acute GvHD at 180 days was similar in patients receiving grafts from donors that were homozygous for the LAG3 rs870849 TT donors had a higher incidence of grades III or IV aGVHD when compared with any other genotype (considering together CC and CT genotypes): 18.6% vs 11.8% respectively .When considering only severe, grades III or IV acute GVHD at 180 days, the incidence was 13.7% for patients receiving grafts from homozygous rs870849 CC donors, 10.3% for CT genotype, and 18.6% for patients with donors homozygous for the T allele (p: 0.082). Patients transplanted from LAG3 rs870849 genetic groups .The incidence of chronic GvHD was similar between the LAG3 rs870849 T allele in the donor was associated with a significant reduction in overall survival at 5 years. This association was dependent of the number of T alleles of the donor  (LAG3 rs870849 TT donors had worse OS than those transplanted from donors with any other genotype (pooling together CC and CT genotypes) . Multivariate analysis using Cox proportional hazard model confirmed that the LAG3 rs870849 TT genotype was an independent risk factor for worse OS .The presence of a LAG3 rs870849 genotype of the donor   . Multivariate analysis with regression modeling of competing risks also failed to detect an association between the LAG3 rs870849 genotype and relapse incidence .Concerning the relapse incidence at 5 years, we did not find statistically significant differences related to the LAG3 rs870849 CT/TT genotype of the donor was not associated with relapse .We performed a specific subgroup analysis in patients with acute myeloblastic leukemia (AML), and we detected that the LAG3 rs870849 genotype of the donor was associated with relapse in patients with or without acute GVHD but did not find this association neither in patients with aGVHD (p: 0.350) nor in those not presenting this complication (p: 0.634).Also, we explored whether PDCD1 genotype of the donor has been previously described as having an impact on clinical outcome after HLA-identical sibling donor transplant, we explored the additive effect of the LAG3 rs870849 C>T and the PDCD1 rs36084323 A>G genotypes of the donor on the severe acute GVHD incidence.LAG3 is stored together with PDCD1, and its liberation seems to have synergistic action by inhibiting T-lymphocyte activation. As the LAG3 rs870849 TT genotype was associated to the PDCD1 rs36084323 GG  or AG  genotypes. The LAG3 rs870849 CC genotype was associated to the PDCD1 rs36084323 GG genotype in 249 cases (34%), AG in 10 cases (1.4%) and AA in 5 cases (0.7%). The LAG3 rs870849 heterozygous CT genotype was associated to the PDCD1 rs36084323 GG genotype in 328 cases (44.8%), AG in 16 cases (2.2%) and AA in 1 case (0.1%).The genotype at both SNPs was available for 732 donors. The LAG3 rs870849 TT genotype was associated with an increased risk of severe acute GVHD independently of the PDCD1 rs36084323 genotype. According to this data and considering the previously reported results considering the clinical outcome in basis to the PDCD1 genotype of the donor, we defined three genetic groups in 741 patients: group 1) 132 patients transplanted from donors homozygous for the LAG3 rs870849 T allele, independently of their PDCD1 rs36084323 genotype; group 2) 577 patients transplanted from donors genotyped as LAG3 rs870849 CT or CC and PDCD1 rs36084323 GG [as the G allele in homozygosis was previously suggested to be associated to a higher incidence of acute GVHD , patients with donors with any other LAG3 genotype but homozygous for the PDCD1 rs36084323 G allele had an intermediate incidence (12%) whereas none of the 30 patients whose donors had LAG3 rs870849 CT or CC and PDCD1 rs36084323 AG/AA genotypes developed grades III to IV acute GVHD (0%). These differences were globally statistically significant (p: 0.025) (LAG3 rs870849 TT genotype and those transplanted from donors genotyped as LAG3 rs870849 CT or CC and PDCD1 rs36084323 AG or AA (p: 0.016), whereas no differences were detected when comparing any of these groups with patients transplanted from donors genotyped as LAG3 rs870849 CT or CC and PDCD1 rs36084323 GG (p: 0.075 and p: 0.057 respectively). Multivariate analysis confirmed that LAG3 rs870849 TT genotype of the donor was an independent risk factor for a higher incidence of acute GVHD, independently of the PDCD1 genotype .When determining the incidence of grades III to IV acute GVHD at 180 days according to these genetic groups we found that patients whose donor was homozygous for the LAG3 rs870849 TT genotype had poorer outcome than those with other genotypes at this SNP and with PDCD1 rs36084323 GG genotype, whereas the minority group with LAG3 rs870849 CT or CC and at least an A allele at PDCD1 rs36084323 had an excellent OS   or group 3 (p: 0.025), whereas direct comparison between groups 2 and 3 did not show differences (p: 0.132). Multivariate analysis including the PDCD1 genotype identified the donor LAG3 rs870849 TT genotype as a risk factor for worse OS .Regarding overall survival, patients receiving grafts from donors with LAG3 rs870849 TT genotype of the donor as an independent risk factor for worse DFS .As expected, DFS followed a similar pattern   or LAG3 rs870849 CT or CC and PDCD1 rs36084323 AG or AA (3.8%). These differences were statistically significant (p<0.001) , whereas the PDCD1 rs36084323 of the donor showed no statistical association .When exploring TRM according to these genetic groups, we found that patients transplanted from donors homozygous for the Many studies have explored the correlation between genetic variations in genes involved in the inhibition of the T-cell immune response and the development of autoimmune diseases. Moreover, genetic polymorphisms in CTLA-4 and PD-1 genes have been also associated with the incidence of graft-versus-host disease after alloHSCT –5.In the present study, we have examined the relationship between the LAG-3 rs870849 genotype of the donor and the clinical outcome after alloHSCT from HLA-identical sibling donor. Our findings show that patients transplanted from donors homozygous for the T allele at the LAG-3 rs870849 SNP have an increased risk of severe acute GVHD, increased TRM and worse survival. These results agree with the previously described association of the T allele with multiple sclerosis, suggesting that the presence of the T allele impairs the inhibitory potential of the LAG-3 molecule in the allogeneic transplant setting, leading to an increased T-cell response.It has been speculated that the rs870849 Isoleucine/Threonine (Ile/Thr) substitution would alter the conformational and functional properties of the protein, reducing the binding affinity of LAG3 and leading to an enhanced expansion of T-cells . HoweverIn vitro, the FcgammaRIIB 232Thr showed to be significantly less potent than wild type 232Ile in inhibiting B cell receptor signaling. Moreover, FcgammaRIIB 232Thr was shown to be less effectively distributed to detergent-insoluble lipid rafts, probably due to the substitution of a large hydrophobic amino acid (isoleucine) by a polar one (threonine) (The relevance of this polymorphism could be explained by looking at the effects driven by the same amino acid polymorphism that has been described within the B cell inhibitory receptor FcgammaRIIB (FCGR2B) gene (rs1050501: FCGR2B c.695T>C) leading to the same amino acid change (Ile/Thr) in the transmembrane helix . In vitrreonine) .In vitro studies to correlate the rs870849 genotype with protein expression are currently in development.However, if this hypothesis is true, the rs870849 T allele (coding for Isoleucine) should be associated with a higher inhibitory activity of LAG3 and therefore with a lower incidence of acute GVHD in the allogeneic model, which is just opposite to our findings. This inconsistence is difficult to explain. One possible explanation would be a different kinetics of proteolytic cleavage of surface LAG3 mediated by disintegrin and metalloproteinase ADAM10 and ADAM17. It has been reported that LAG-3 and PD-1 molecules have a synergistic effect on T-cell inhibition . We explThis observation may be relevant, as cancer immunotherapy based on immune checkpoint inhibitor blockades is currently one of the main therapeutical approaches for several neoplasms. It is well known that PD-1/LAG-3 expression is a marker of exhausted T-cells infiltrating tumors , and higLAG-3 genotype of the donor with the occurrence of acute GvHD, transplant-related mortality and overall survival after allogeneic transplant in a large cohort of Caucasian patients.This is the first study correlating the In contrast, a recent Japanese study explored the relationship between transplant outcome and polymorphisms in the immune checkpoint genes, including the LAG3 rs870849 SNP in patients receiving an unrelated alloHSCT, and the authors failed to find any correlation . There aThese results need to be confirmed by analysis of independent patient cohorts, but our approach suggests that the routine determination of polymorphisms in genes coding for immune checkpoint inhibitors should be considered when evaluating the predicted clinical outcome associated for each available donor. The limitations of the present study are that we did not account for multiple comparisons and the associations have not been validated by testing in an independent cohort.The current results could not be applied clinically in donor selection for related alloHSCT, due to the limited availability of HLA-identical siblings, but they potentially could be used in donor selection for unrelated HCT, if these results are reproducible in a similar study with unrelated donors for Caucasian patients.The original contributions presented in the study are included in the article/The study was approved by the Ethics Committee of the Dr. Josep Trueta University Hospital . Written informed consent to participate in this study was provided by the participants’ legal guardian/next of kin.DC and DG designed the research, analyzed data and wrote the article; DC, RR-R, MG-B, NS, NL, PV, ET, and RC performed research, IG-C, RdC, IH, IB, CM, MG, GS, CF, AS, JP, JL-J, and MJ participated in the collection and analysis of data. All authors contributed to the article and approved the submitted version.


Various surgical methods to prevent postoperative cerebrospinal fluid (CSF) leaks during transsphenoidal surgery have been reported. However, comparative studies are scarce. We aimed to compare the efficacy of a fibrin-coated collagen fleece (TachoSil) versus a dural sealant  to prevent postoperative CSF leakage. We perform a retrospective study comparing two methods of sellar closure during endoscopic endonasal transsphenoidal surgery (EETS) for pituitary adenoma resection: TachoSil patching versus DuraSeal packing. Data concerning diagnosis, reconstruction technique, and surgical outcomes were analyzed. The primary endpoint was postoperative CSF leak rate. We reviewed 198 consecutive patients who underwent 219 EETS for pituitary adenoma from February 2007 and July 2018. Intraoperative CSF leak occurred in 47 cases (21.5%). A total of 33 postoperative CSF leaks were observed (15.1%). A reduction of postoperative CSF leaks in the TachoSil application group compared to the conventional technique using Duraseal was observed  although non-statistically significant. Two patients required lumbar drainage, and no revision repair was necessary to treat postoperative CSF rhinorrhea in Tachosil group. Fibrin-coated collagen fleece patching may be a valuable method to prevent postoperative cerebrospinal fluid (CSF) leaks during EETS for pituitary adenoma resection. Endonasal endoscopic transsphenoidal surgery (EETS) has become the preferred method for treating these tumors2. While the procedure is considered safe and effective, cerebrospinal fluid (CSF) leak remains a significant complication after EETS3Pituitary adenomas represent approximately 15% of all intracranial neoplasms9. Autologous grafts can lead to additional incisions, increased operative time, risk of additional complications, and patient discomfort10. To avoid these limitations, cadaveric acellular dermis or cadaveric fascia lata can be used12. Various adhesive substances that locally reinforce sellar repair can also be used, either alone or in combination, including fibrin sealants or collagen-based compounds17. Finally, vascularized flaps have gained increasing popularity10. However, the nasoseptal flap (NSF) is not exempt from complications and can lead to nasal discomfort, excessive crusting, anosmia, and the necessity for multiple debridements9. The ideal alternative to these techniques would be an option effective at preventing CSF leak, technically simple to handle, inexpensive, and minimizing morbidity7.Numerous techniques of sellar reconstruction to prevent this complication have been described. Many involve using autologous tissue grafts, including muscle, septal cartilage/bone, fat, or free mucosal flap4. The preparation is ready to use and can be applied directly to the target tissue4. Recent studies have demonstrated that TachoSil can be useful during EETS, obviating the need for autologous tissue grafts, postoperative lumbar drainage, and NSF reconstruction6.TachoSil  consists of a sheet of collagen, which is coated on one side with human fibrinogen, human thrombin, and riboflavinIn this study, we compare the safety and efficacy of a fibrin-coated collagen fleece (TachoSil) patch with fibrin glue versus a dural sealant  to prevent postoperative CSF leakage.The Erasmus-ULB ethics commitee approved this study (P2018/408) and waived patient consent due to the retrospective study design. Eligible patients were identified from a prospectively maintained institutional pituitary tumor database. All procedures were performed by senior authors (SH and ODW) with extensive experience in pituitary surgery . Eligible patients underwent EETS for pituitary adenoma between February 2007 and July 2018. The primary outcome was postoperative CSF leak rate following sellar repair. Secondary outcomes included the lumbar drain use and reoperation for repeat surgical closure. For each patient, a thorough chart review was conducted, and information obtained included: patient age, gender, tumor size, modified Hardy’s classification for sellar invasion and suprasellar extension, previous surgery, histopathologic diagnosis, intraoperative CSF leakage, repair method, postoperative complications, management of postoperative CSF leak and the length of stay.Under general anesthesia, the patient was placed in a supine position. The approach was performed via one nostril. A 30° rigid endoscope was introduced into the nasal cavity and the middle turbinate was lateralized by gentle pressure with an elevator. The rostrum was partially resected. An anterior sphenoidotomy was performed, and the sphenoid septum was removed. Medtronic electromagnetic neuronavigation system was used to tailor precisely the exposure to the tumor size. After complete tumor removal, the sellar cavity was then explored for evidence of a CSF fistula or tumor remnant. Two different methods were successively used to reconstruct the sella. Accordingly, the patients were divided into cohorts based on the closure technique. In cohort 1 (from 2007 to 2015), we packed the sphenoid sinus with a dural sealant  alone. In cohort 2 (from 2015 to 2018), we covered the anterior wall of the sella by a single layer of the TachoSil patch and then packed the sphenoid sinus with a fibrin sealant .Intraoperative CSF leaks were classified in operative protocols as « low » flow CSF leaks which are small "weeping" leaks flowing even without Valsalva maneuver and “high” flow CSF leaks which are moderate or large CSF leaks with or without obvious diaphragmatic defect. In the instance of an intraoperative CSF leak, the postoperative cavity dead space was usually filled with a small piece of absorbable gelatin foam  to reduce the amount of CSF leak. For “low” flow CSF leaks, the closure was then performed as described in the surgical technique for each cohort. For “high” flow leaks, an additional covering by an autologous tissue  of the sellar opening was used before classical closure for each cohort. Before 2015, we used in our practice a conventional sellar closure technique with the dural sealant closure . In 2015, as we aimed to reduce the incidence of postoperative CSF leaks, we opted for a promising alternative method using a fibrin-coated collagen fleece (TachoSil).All patients were admitted to the intensive care unit (ICU). On the first postoperative day, patients were transferred to the ward if they were clinically stable and did not present complications.p was < 0.05. All statistical tests were two-sided and performed using IBM-SPSS version 26.0 software  and MedCalc Statistical Software version 14.12.0 .Continuous variables are summarized by means and standard deviations (SD) and qualitative variables as numbers and percentages. Differences in continuous variables means between two groups were compared using classical Student t-tests or Welch's t-tests in case of variance inequality. Differences in qualitative variables were compared between groups using Pearson's exact chi-square tests. Statistical significance was considered when For this type of study informed consent was waived with the acceptance of ULB-Erasmus university hospital’s ethic committee.All methods were carried out in accordance with relevant guidelines and regulations of the 'Declaration of Helsinki'.A total of 219 EETS for pituitary adenoma resection were performed on 198 patients at our institution between February 2007 and July 2018. Twenty patients underwent repeat transsphenoidal surgeries for recurrent or residual adenoma. Nineteen patients underwent two operations and one patient three operations. Sellar closure with TachoSil was applied in 65 surgeries, and conventional packing with DuraSeal was applied in the remaining 154 surgeries , and no patient required revision repair surgery (0% versus 2.6%) without significance.EETS has become the preferred method for the treatment of many pituitary adenomas1. In this context, our findings are more striking and may suggest that Tachosil closure has greater efficacy at preventing CSF leak than Duraseal packing alone. Other predictors of postoperative CSF leak, such as intraoperative CSF leak, tumor size, and repeat surgery, were not significantly different between the cohorts2.Interestingly, there were significantly more patients with suprasellar extension in the Tachosil cohort, a known predictor of postoperative CSF leaks2.TachoSil has several potential advantages for sellar reconstruction when compared to other techniques. First, it serves as a reliable barrier by attaching firmly to dura. Its placement can be performed in a narrow field with straightforward surgical handling. From our experience, it can be easily removed in recurrent tumor surgery. Finally, it provides hemostasis with antigenicity—a feature associated with a lower incidence of postoperative infection4. In another study performed on 19 grade 3 intraoperative CSF leaks, postoperative CSF leakage following Tachosil repair utilizing the "sandwich technique" was 5.3%2. Tamasauskas et al. performed a sellar closure using Tachosil and Surgicel  in 29 patients who underwent EETS, and none had a postoperative CSF leak6. Currently, the "sandwich technique" using Tachosil appears more effective at preventing postoperative CSF leaks than a single layer technique, which was utilized in our study.While our study found fewer postoperative CSF leaks in the Tachosil cohort, it remains higher than other studies using the same material for sellar closure Table . A possi5. Classically, surgeons have used autologous materials such as abdominal fat, muscle, or free mucosal graft with or without support for the graft from nasal bone or cartilage to prevent postoperative CSF rhinorrhea9. These methods are effective for this purpose, with a rate of postoperative CSF leaks ranging from 0 to 10%, similar to our study17. While these grafts have the advantage of compatibility and cost, harvesting them can prolong the operative time and often requires a separate surgical incision, which can also be associated with complications  and cause additional discomfort to patients7. Fat may also interfere with the interpretation of the sellar content on postoperative MRI3. Inadequate packing may aggravate the arachnoid tearing and compress the optic chiasm5. These limitations are partially addressed by using cadaveric materials  for sellar floor reconstruction in transsphenoidal surgery  and 6.7% (19 of 283), respectively18. In a series of 151 patients with intraoperative CSF leaks of whom 144 received Hadad-Bassagestaguy nasoseptal flaps, only 3.3% developed postoperative CSF leak , and Floseal . Nevertheless, our results suggest a significantly higher rate of postoperative CSF leak when using Duraseal alone.Vascularized flaps are currently thought to be the most effective technique for endoscopic endonasal reconstruction17. For example, Zhou et al. have demonstrated that only 6 of the 492 (1.2%) cases using a graded repair method subsequently developed postoperative CSF leak  and those occurring through the sealant (Tachosil)23. These results could explain why "the sandwich technique," using two layers of Tachosil, seems superior to the single-layer technique, as it prevents the second time of the trans-graft leak.In a laboratory study, Chauvet et al. found the mean pressure at which a leak visually occurred with Bioglue , Duraseal, Tachosil and Tissucol  was 16.78, 28.31, 27.09 and 10.03 mmHg, respectively, which suggest that Duraseal and Tachosil may be superior for closureBesides its retrospective design, the main limitation of this study is the sequential use of the two different techniques investigated . One could expect that experience gained during the first part of the study  may lead to bias in favor of the last technique used (i.e. Tachosil). However, this potential bias is limited by the extensive prior experience of the operators—although mainly microscope-assisted pituitary surgeries.Fibrin-coated collagen fleece patching may be a valuable method to prevent CSF leaks during EETS for pituitary adenoma resection. This study reports fewer postoperative leaks in the TachoSil cohort compared to the Duraseal cohort without reaching significance. This observation dovetails with previous surgical series and experimental data but powered studies to achieve higher levels of evidence are required to confirm these results.


BackgroundThe morbidity of the donor site in split-thickness skin graft (STSG) may include abnormal pigmentation, delayed healing, and unfavorable scarring. Studies are usually focused on improving the healing of the recipient site, so donor site management becomes a secondary consideration. An optimal solution should be sought for donor site management to improve healing and minimize morbidity.MethodsIn this study, we used minced residual skin grafts over half of the donor site (cases) and compared the healing duration and scar quality with the other half (control). Healing duration was measured in days and the scar quality was assessed by the Patient and Observer Scar Assessment Scale (POSAS) at 90 days, 180 days, and 360 days.ResultsThe healing time was reduced with the application of minced residual skin grafts on the donor site. The scar quality was significantly better in the case group as compared to the control group at 90 days, 180 days, and 360 days (p<0.05).ConclusionMincing residual skin grafts and replacing them back to the donor site reduces the healing time and improves the quality of the scar. Split-thickness skin grafting (STSG) is one of the most extensively performed procedures to achieve wound closure . Most stDonor site morbidity of STSG is usually minimal, but that may not always be the case . MorbidiSmall portions of the skin graft left at the end of the procedure or the graft obtained after trimming the edges are usually discarded. There have been only a few studies demonstrating the benefit of the minced residual skin graft on the donor site, resulting in earlier epithelialization and also improving the appearance -8. With This article was previously presented as an oral presentation at the 29th Annual Conference of the National Academy of Burns of India- NABICON 2021-22 on January 22, 2022.Aims and objectivesThe aim of this study is to assess the efficacy of minced residual skin grafts in improving donor site healing  and scar quality . The variables used to study the above-mentioned objectives were: (i) time taken to epithelialize the entire donor site (as assessed by observers) or duration of healing - expressed in number of days; and (ii) scar parameters at 90 days (three months), 180 days (six months), and 360 days (one year) as assessed by POSAS.POSAS is a validated scar assessment scale that measures scar quality by evaluating visual, tactile, and sensory characteristics of the scar from two different perspectives: the patient perspective and the surgeons’ perspective . The patThe authors assert that this study complied with the ethical standards of relevant national and institutional guidelines on human experimentation and the Helsinki declaration of 1975 (revised in 2008). Informed consent was obtained from all patients. This is an open-label, prospective, case-control study conducted for a period of one year (March 2020 to February 2021).Inclusion and exclusion criteriaAll patients who were posted for STSG and gave consent to participate in the study were included in the study. Exclusion criteria were as follows: (i) patients under the age of 18 years, (ii) patients with a known tendency for hypertrophic scarring/keloids, (iii) pregnancy, and (iv) co-morbid factors like uncontrolled diabetes, ischemic heart disease (IHD), renal failure, use of steroids. After evaluation of patients by the inclusion and exclusion criteria, suitable patients (n=30) were included in the study.Surgical techniqueGraft harvest was done under strict aseptic conditions using Watson's modification of Humby's knife with the graft thickness set at 0.15-0.30 mm. The size of the graft harvested was decided based on the wound requirements. The graft was secured over the wound, and the excess graft from the wound edges was trimmed. The residual graft was thoroughly washed with saline to prevent donor site contamination. These trimmed graft pieces, along with any unused grafts, were minced manually with blades and scissors into approximately 1 mm pieces. The donor area was divided into two approximate halves based on the number of 10 cm × 10 cm tulle dressings needed to cover the raw area. Hemostasis of the donor site was achieved, followed by the application of minced skin grafts.In the case group, the minced skin graft was applied evenly over one-half of the donor site and covered with an occlusive non-adherent dressing in the form of a tulle and polyurethane foam, followed by an absorbent gamjee roll.In the control group, the remaining half of the donor site was dressed with an occlusive non-adherent dressing alone in the form of a tulle and polyurethane foam, followed by an absorbent gamjee roll.Postoperative managementAfter every dressing change session, photographic documentation was made. In the early postoperative period, the donor site dressing was assessed for soakage. If the dressing appeared soaked, the gamjee roll was gently replaced without disturbing the tulle and polyurethane foam dressing. Also, the donor site dressing was regularly assessed to see if there were signs of loosening, which was indicative of underlying epithelialization.In our practice, we noticed that the donor sites were usually epithelialized by postoperative day (POD)-14 and that the dressings would slide off the donor sites on their own. With our intervention, we wanted to assess whether the application of minced skin grafts led to an earlier epithelialization. If the donor site dressing did not come off on its own, the first dressing change was done on POD-12 and subsequently every alternate day till complete epithelialization was noted in each group. The duration of healing was defined as the number of days required to achieve complete epithelialization.The patients were followed up at 90 days (three months), 180 days (six months), and 360 days (one year) to assess for scar quality using the POSAS score. Patients were advised twice a day application of coconut oil or an emollient followed by a massage for 10 minutes (once in the morning and once in the evening), once the donor site had epithelialized.Data analysisDuration of healing, POSAS score, and other demographic parameters were recorded and analyzed using the OpenEpi calculator available online . DuratioDemographic characteristicsOur study included 21 males and 9 females. The average age of our study was 36.6 years. The average age of males was 43.2 years (range 18-67), whereas the average age of females was 30.8 years (19-50).Duration of healingA statistically significant difference  in the mean duration of healing time was noted for both groups. Healing time for the intervention/study group was shorter (14.93 ± 1.79 days with a range of 12-20 days) compared to the control group (16.13 ± 2.09 days with a range of 14-22 days).Scar qualityScar quality evaluation was assessed using POSAS at 90 days (three months), 180 days (six months), and 360 days (one year) from the date of surgery.At 90 days, the mean in the case group when compared against the control group revealed lower values in both the observer  and the patient scales  with a 'p'-value of 0.02 and 0.03, respectively. Similarly, at 180 days, comparison of the mean in the case group against the control group revealed lower values in both the observer  and the patient scales  with a 'p'-value of 0.03 and 0.02, respectively. The same trend was noticed at 360 days, with a lower mean seen in the case group as compared to the control group in both the observer  and the patient scales  with a 'p'-value of 0.02 and 0.03, respectively.The POSAS score in the case group was significantly lower than that of the control group at three months, six months, and one year on the patient as well as observer scales, suggesting that there was an improvement in scar quality in the case group as compared to the control group and that the finding was statistically significant Figure .Historically, skin graft donor sites have been managed with different kinds of specialized dressings ,3. Such The concept of transplanting small parts of the skin onto wounds was probably first described by Reverdin as "pinch grafting" . In thisWe used the technique of mincing the unused and residual graft pieces trimmed from the edges of the recipient site. The duration of healing was significantly less in areas where the minced residual graft was applied than in areas where no graft was applied. Mincing led to a faster rate of epithelialization, possibly due to the release of keratinocytes, melanocytes, and stem cells in an environment of growth factors ,8,17. ThThe improved appearance and reduced itchiness/pruritis at the donor site may be due to the release of growth factors such as interleukin-6 (IL-6), granulocyte-colony-stimulating factor (G-CSF), monocyte chemoattractant protein-1 (MCP-1), vascular endothelial growth factor (VEGF), and platelet-derived growth factor (PDGF) . This maMincing the excess graft and replacing it on the donor sites reduced the time to heal and improved the scar quality Figures -6. This The excess grafts at the recipient site edges are either left or trimmed and discarded. So, this technique provides a win-win situation. The small graft pieces that otherwise would have been wasted or discarded are put to use in improving the donor site healing. The benefits or advantages of minced residual skin grafting have been shown in Figure The possible disadvantages of minced residual skin grafting might be that the procedure is more time-consuming as compared to performing no grafting. There is a risk of transmission of infection/contamination from the recipient site to the donor site. This can be prevented by thorough washing with saline before applying the residual grafts onto the donor site. Also, sometimes it may not be possible to cover the entire donor site with the residual minced grafting. We feel that despite these possible drawbacks, the advantages of the procedure far outweigh the disadvantages. As more studies contribute to the research, minced residual skin grafting can be advocated as a routine procedure following STSG.Our study does have a few limitations. The sample size of our study was small. In our study, the available minced grafts were spread evenly over the case area without any particular ratio being observed. A further study with different ratios of spread might help in determining the optimum ratio, if any, to achieve faster healing. Also, there was no standardization of the control versus study site selection except for partitioning into two similar halves.The donor site of STSG treated with minced residual skin grafts showed a reduction in healing time and the healed scar had a significantly better appearance as compared to the donor site treated without a skin graft. Our study shows that, when applicable, this technique can be incorporated as a "standard procedure" following STSG to achieve optimal wound healing of the donor site.


Several polymeric blends based on poly[1-trimethylsilyl-1-propyne] (PTMSP) and poly[vinyltrimethylsilane] (PVTMS) were studied. The gas and water vapor sorption and permeability coefficients for the PTMSP/PVTMS blend membranes at different PVTMS contents (0–100%) were obtained under temperatures of 30 and 60 °C for the first time. As the PVTMS content increases, the O2 and CO2 permeabilities decrease by 160 and 195 times at 30 °C, respectively. The fractional accessible volume of the polymer blends decreases accordingly. The transport of the CO2 capture solvent vapors through the PTMSP/PVTMS blend membranes were determined in thermo-pervaporation (TPV) mode using aqueous monoethanolamine (30%), N-methyldiethanolamine (40%), and 2-amino-2-methyl-1-propanol (30%) solutions as model amine solvents at 60 °C. The membranes demonstrated high pervaporation separation factors with respect to water, resulting in low amine losses. A joint analysis of the gas permeabilities and aqueous alkanolamine TPV data allowed us to conclude that the polymer blend composition of PTMSP/PVTMS 70/30 provides an optimal combination of a sufficiently high oxygen permeability and the pervaporation separation factor at a temperature of 60 °C.In this work, perspective polymeric materials were developed for membrane contactor applications, e.g., for the dissolved oxygen removal from amine CO PTMSP is shown to be promising as a selective layer material for composite membrane contactors designed for a CO capture ,29,30. T0 barrer ), thus t2 desorption from an amine solvent [Recent studies on the membranes from the polymer blends have demonstrated the potential of a blending approach for a “fine-tuning” of the fractional free volume (FFV) of the polymeric membrane materials, resulting in a tailoring of their transport properties . In part solvent . However3 were used. Monoethanolamine ), N-methyldiethanolamine ), and 2-Amino-2-methylpropane-1-ol ) were used for the preparation of the model amine solvents. Carbon dioxide, nitrogen, and oxygen  were used for the permeability and sorption measurements.A commercial polymer PTMSP ) and a commercial PVTMS sample with a density of 0.86 g/cm® 273 Quick Step  electron micrometer with an accuracy of ±2 microns.The properties of polymer composite materials PTMSP/PVTMS were studied using homogeneous dense membranes, which are films with a thickness of 25–45 microns. A number of PTMSP/PVTMS solutions were prepared see  by mixin3).The value of the fractional accessible volume (FAV) of the composite polymer materials was determined by the extended hydrostatic weighing method according to the methodology presented in . The met2, CO2, and N2. A manometric method for determining P using an automatic high-precision setup  was used. The effective area of the samples was 13.8 cm2. The experiments were carried out at temperatures of 30.0 ± 0.1 °C and 60.0 ± 0.1 °C (the maximum temperature of a carbonized amine solvent at the outlet of the absorption column in the CO2 capture system) at a gas pressure supplied to the membrane of 0.2–0.8 bar. Before measuring, the films were kept at these temperatures for at least 2 h to stabilize the transport properties. The gas permeability coefficient (Pi) was expressed in barrers (1 barrer = 10−10 Ncm3·cm/(cm2·s·cm Hg.) and was determined by a linear extrapolation of the experimental data to zero the transmembrane pressure value. The ideal selectivity of the films was determined by the ratio of the permeability coefficients of individual gases (α = P1/P2).The transport properties of the polymer blend membranes were determined by measuring the gas permeability coefficients (P) of the films for pure gases: O2, CO2, and H2O were measured using the XEMIS-002 gravimetric sorption analyzer  at temperatures of 30 and 60 °C and pressures up to p = 1 atm (for gases) or up to p/p0 > 0.75 (for water).The sorption isotherms of OBefore starting the measurement, the film samples were vacuumed at 60 °C for 24 h to remove the solvent residues. Further, by the method of a helium calibration, described in detail in the work of , the skeEach sorption isotherm was obtained at least 3 times to control the convergence of the results. Between measurements, the sample was vacuumed for 2 h at the temperature of the experiment. According to the sorption kinetics, the time for the mass of the sample to reach a stationary value did not exceed 20 min.Calorimetric studies were performed in argon with calorimeter Mettler DSC823  at a scanning rate of 10 deg∙min−1 in the range of 140–100 °C. The glass transition can be observed as the midpoint of a step in the baseline of the measurement DSC curve.Scanning electron microscopy (SEM) was used to characterize the structure and morphology of the polymer blend membranes. SEM was carried out on a Thermo Fisher Phenom XL G2 Desktop SEM . Cross-sections of the membranes were obtained in liquid nitrogen after a preliminary impregnation of the specimens in isopropanol. A thin (5–10 nm) gold layer was deposited on the prepared samples in a vacuum chamber (≈0.01 mbar) using a desktop magnetron sputter, the Cressington 108 Auto Sputter Coater . The accelerating voltage during the image acquisition was 15 kV.2 removal process from the amine solvents in the gas–liquid membrane contactor, the competitive transport of solvent vapors along with the targeted transport of O2 molecules is inevitable. In this regard, the transport properties of the polymer blend membranes toward solvent vapors were studied. For this purpose, the thermo-pervaporation (TPV) of pure aqueous amine solutions  was carried out. All the solutions were prepared from pure alkanolamines and distilled water by the weight method. The experiments to study the properties of the polymer blend membranes were carried out on the lab-scale unit shown in 2. The TPV process was carried out at T = 60 (±0.2) °C, while the pressure in the submembrane space was equal to the atmosphere. The permeate was condensed at Tcond = 10 °C. The width of the air gap was 2.5 mm. The total permeate flow J, kg/m2·h, was determined by the method according to the Formula (1).2) during t, h. The separation factor was determined by the Formula (2).ox and wx are the mass fractions of alkanolamine and water in the separated mixture, and oy and wy are the mass fractions of alkanolamine and water in permeate.During the O3), equipped with an EMCES 21 electric suppressor and a conductometric detector CD-510 with an electrical conductivity measurement accuracy of 0.1%.Permeate and retentate were analyzed by ion exchange chromatography using the “Aquilon-Stayer-Ì”  system  eluent, an aqueous solution of nitric acid with a concentration of 0.004 mol/dmThe properties of pure PTMSP and PVTMS materials and their blends were studied using SEM and DSC. It was revealed that with a complete uniformity of pure PTMSP and PVTMS films, their blends exhibit a more complex delaminating structure, especially at a higher PVTMS content. At the same time, it is clearly noticeable that with an increase in the PVTMS content, the nature of the resulting structures changes. Most likely, this change in the morphology is caused by the phase separation of the polymers. Based on the results of the DSC, a set of curves presented in 2, N2, and O2) permeability through dense polymer blend films, measured at 30 °C and 60 °C, are presented in The data on single gases v et al.  estimateThe observed deviations of the gas permeability coefficients from the linear dependence in the logarithmic scale correlate with the SEM results and indicate the phase separation of the polymers. These differences may be associated with the polydispersity of polymer samples, that is, with a non-monomodal distribution of the polymer molecular weights.2, CO2, and N2 for PTMSP are 9000, 35,700, and 6000 barrer, respectively. This is approximately 160, 195, and 430 times higher than the permeability values for PVTMS, respectively. Thus, the PTMSP and PVTMS blends are a convenient material for creating membranes with a wide range of gas permeability variations. In turn, the change in the dependence of the gas permeability coefficient on the composition of the PTMSP/PVTMS blend demonstrates a higher permeability for the blends up to 30% and a sharper drop after  from 5 to 9, while α(CO2/O2) decreases from 3.1 to 2.5.It is known that for such highly permeable glassy polymers such as PTMSP, the “physical aging” effect is especially pronounced. At the same time, there is a significant decrease in the gas permeability over time . An incrIt is known that the transport of gases in polymer membranes is determined by the fractional free volume of the polymer. In this regard, the values of fractional accessible volume (FAV) of the PTMSP/PVTMS blends were determined, as shown in 2 and CO2 in pure PTMSP and PTMSP/PVTMS blends are shown in 2 sorption (2 than blend #3 with 80 wt% of the PVTMS content.The sorption isotherms for Osorption . The samS) for both gases are given in S values decrease as the fraction of PVTMS in its blends with PTMSP increases, and as the temperature rises. The decrease in the gas solubility is associated with the compaction (densification) of PTMSP when blended with PVTMS [2 and O2, i.e., a so-called ideal solubility selectivity, varies non-monotonically with an increasing PVTMS content in the blend PTMSP/PVTMS. This ratio slightly decreases at a 30% PVTMS content and then increases to values of about 9 at an 80% PVTMS content in the blend  was described by the modified BET equation [Water vapor sorption equation , also caequation ):(4)V=VmmV is the monolayer sorption capacity, K is the binding constant describing the sorbate–sorbent interaction, and asK is the binding constant describing the sorbate–sorbate interaction in the side associate. The calculated parameters are listed in Here, p/p0 < 0.2 and almost independent of PVTMS content in the samples. At p/p0 > 0.2, the water sorption in PTMSP increases sharply, in contrast to the samples containing PVTMS, and reaches values comparable to and even exceeding the CO2 sorption at the same temperature  > AMP (1.1–4.8 kg⋅µm⋅m−2⋅h−1) > MDEA (0.8–4.0 kg⋅µm⋅m−2⋅h−1). These results can be explained by the corresponding drop in the saturated vapor pressures of the studied amines (see for example [Since the membrane contactor which should be applied to remove dissolved O example ), and an2 removal from the amine solvents. This composition provides an optimal combination of sufficiently high oxygen permeability values and the amine/water separation factor at 60 °C (the maximum temperature of carbonized solvent at the outlet of the absorption column). Thus, it can be expected that a composite membrane with a selective layer from blend #4 will allow for an effective deoxygenation of the solvent in a membrane contactor with insignificant losses of alkanolamine.It is noteworthy that the separation factor increases most strongly during the transition from the PTMSP to blend #4 b. Its va2 solvents in gas–liquid composite membrane contactors have been studied. The permeability of O2, CO2, and N2 through dense films of the PTMSP/PVTMS blends with the PVTMS content was studied at 0, 10, 20, 30, 40, 60, 80, and 100% at temperatures of 30 and 60 °C. As the PVTMS content in the blend increases, the O2 and CO2 permeability coefficients at 30 °C decrease by 160 and 195 times, respectively. It is shown that the form of the dependences of the permeability coefficients of the studied gases on the PVTMS content in the PTMSP/PVTMS blend is close to linear in the logarithmic scale in the range from 0% to 30% and in the range from 30% to 100%, however, there are differences in the form of dependence for these ranges. In particular, a sharper decrease in the permeability coefficient was observed for the zone after 30%. These results are also consistent with the visual representation in the microphotographs. The results on the gas permeability are consistent with the values of the fractional free volume of the polymer blends. Thus, the addition of PVTMS leads to a monotone decrease in the FAV value from 26.5% for pure PTMSP to 15.7% for a blend of PTMSP/PVTMS of a composition of 20/80. The PTMSP/PVTMS blends are a convenient material for creating membranes with controlled characteristics in a wide range of gas permeability variations.The dense membranes made of blends of the glassy polymers PTMSP and PVTMS perspective as selective layers for the deoxygenation of alkanolamine CO2 permeability decreases by 2.6 times from 9000 to 3400 barrer. At the same time, an increase in the temperature leads to an increase in the gas permeability coefficients for PVTMS. As a result, at 60 °C, an increase in the PVTMS content in the blend leads to a decrease in the ideal selectivity of α(CO2/O2) from 3.1 to 2.5. This should have a positive effect on the competitive transfer of CO2 compared to O2.As a result of the strong physical aging effect for PTMSP, its gas permeability coefficients at 60 °C are noticeably lower than at 30 °C. The coefficient of the O2, CO2, and water vapor were measured in pure PTMSP and the PTMSP/PVTMS blends 70/30 and 20/80 at 30 and 60 °C. Unlike the water vapor sorption, gas sorption isotherms are linear in nature. An increase in the temperature from 30 to 60 °C leads to a decrease in the gas solubility coefficients, which is consistent with the data on gas permeability. The isotherms of the water vapor sorption are nonlinear in nature and can be described by the modified BET equation.The sorption isotherms of O2 capture amine solvents in gas–liquid membrane contactors.The transport properties of membranes based on the PTMSP/PVTMS blends toward solvent vapors were determined in the thermo-pervaporation mode using aqueous solutions of MEA (30%), MDEA (40%), and AMP (30%) at a temperature of 60 °C. The membranes demonstrated high pervaporation separation factors for water, which suggests negligible amine losses during the deoxygenation of solvents in the membrane contactor. A joint analysis of the TPV data and gas permeabilities of the polymer blends allowed us to conclude that the composition of PTMSP/PVTMS 70/30 provides an optimal combination of sufficiently high values of oxygen permeability and the pervaporation separation factor at 60 °C. Thus, it can be expected that the PTMSP/PVTMS 70/30 blend is a promising material for the development of new composite membranes designed for the deoxygenation of CO