Sequential Monitoring Research Articles

A Scalable Approach to Assess the Safety of Recently Marketed Systemic Treatments for Atopic Dermatitis in Clinical Practice: First Analysis Cycle of a Sequential Monitoring System

Targeted systemic immune-modulating drugs (IMDs) to treat atopic dermatitis (AD) were highly efficacious in randomized trials. Trials with limited number of subjects leave questions about their safety. We describe a data and analytics structure for the production of timely, high-quality evidence on the comparative safety of recently approved IMDs in patients with AD in clinical practice.We established a series of sequential propensity score (PS)-balanced cohorts that grow in size with each annual data refresh. Nine health outcomes of interest plus conjunctivitis as a positive tracer outcome were identified. The initial treatment comparison was dupilumab, an interleukin-4/13 inhibitor, or tralokinumab, an interleukin-13 inhibitor, versus abrocitinib/upadacitinib, both JAK inhibitors.The first analysis cycle (December 2021-February 2023) compared 269 patients initiating JAK inhibitors and 2,650 initiating IL-4/IL-13 inhibitors. Patient characteristics were well balanced after PS-matching. Outpatient infections within 180 days occurred in 18% of JAK-1 inhibitor initiators versus 12% of dupilumab/ tralokinumab initiators (RR=1.50; 0.96 to 2.33) whereas acne risks were 7% vs. 3%, respectively (RR=2.29, 0.96 to 5.46).This sequential monitoring system will produce essential knowledge on the safety of IMDs to treat AD based on its growing study size of patients observed in clinical practice.

The Role of Digital Dermoscopy and Follow-Up in the Detection of Amelanotic/Hypomelanotic Melanoma in a Group of High-Risk Patients-Is It Useful?

The prognosis, outcome, and overall survival of melanoma patients improve with early diagnosis which has been facilitated in the past few decades with the introduction of dermoscopy. Further advancements in dermoscopic research, coupled with skilled, educated dermatologists in dermoscopy, have contributed to timely diagnoses. However, detecting amelanotic and hypomelanotic melanoma remains a challenge even to the most skilled experts because these melanomas can mimic inflammatory diseases, numerous benign lesions, and non-melanoma skin cancers. The list of the possible differential diagnoses can be long. Melanoma prediction without the pigment relies only on vascular criteria, and all classic dermoscopic algorithms have failed to fulfill our expectations. In fact, the diagnosis of amelanotic and hypomelanotic melanomas is very challenging, which is why every tool in detecting these lesions is of significance. This review aims to explore the current knowledge and the literature on the possibility of detecting amelanotic/hypomelanotic melanomas using sequential monitoring with digital dermoscopy and total body skin photography.

An efficient multivariate depth-based EWMA control chart for monitoring location

In multivariate statistical process control (MSPC), when there is a limited knowledge or there is no knowledge about the underlying process distribution, nonparametric control charts are useful. This paper introduces a new powerful multivariate depth-based control chart for online monitoring of location parameters in MSPC. Actually, by incorporating the exponentially weighted moving average control scheme, the weighted version of a well-known multivariate depth-based test statistic is used to formulate the new charting statistic. The methodology is based on adapting the intended charting statistic to the online sequential monitoring of the location parameters. The proposed control chart is affine-invariant and has a conditional distribution-free property over the class of elliptically symmetric distributions. This control chart is also very efficient in detecting small or moderate shifts in the process location parameter, especially for large dimensions.

A Pilot Study of the CD38 Antagonist Daratumumab in Patients with Metastatic Renal Cell Carcinoma or Muscle-Invasive Bladder Cancer.

We performed a pilot study of daratumumab (an mAb directed against CD38) in muscle-invasive bladder cancer (MIBC) and treatment-refractory metastatic renal cell carcinoma (mRCC). Patients with MIBC underwent baseline transurethral resection of the bladder tumor followed by four weekly doses of daratumumab prior to cystectomy. Patients with mRCC underwent baseline and sequential biopsies after eight weekly doses. The primary endpoint was safety. The secondary endpoints were pathologic complete response rate for the MIBC cohort and objective response rate and progression-free survival for the mRCC cohort. Exploratory analyses included immune monitoring and overall survival. A Bayesian sequential monitoring design for toxicity was used for excessive toxicity. In both the MIBC (n = 8) and mRCC (n = 8) cohorts, no toxicity events were encountered. In the MIBC cohort, one patient experienced pathologic complete response rate. In the mRCC cohort, no objective responses were reported, and the median progression-free survival was 1.5 months (95% confidence interval, 1.1-1.8 months). Immune monitoring found significant reductions in NK cells in circulation in both cohorts after treatment. In the tissue analysis, IHC found evidence of diminished CD38 presence in mRCC with treatment, whereas the baseline levels in MIBC were low. Treatment with daratumumab was safe. No signal of efficacy was detected in mRCC, and conclusions on the activity in MIBC were limited. Evidence of daratumumab targeting CD38 was detected in circulating immune cells and within the tumor microenvironment of mRCC and MIBC. In this prospective clinical trial of daratumumab, treatment in patients with MIBC and mRCC was safe. Limited efficacy was observed. Treatment with daratumumab resulted in CD38-expressing immune cell subsets to be targeted both in circulation and within the tumor microenvironment.

Oral sulfate solution versus polyethylene glycol for bowel preparation before colonoscopy, meta-analysis and trial sequential analysis of randomized clinical trials.

This study aimed to compare oral sulfate solution (OSS) with polyethylene glycol (PEG) for bowel preparation before colonoscopy. A literature search was performed on PubMed, Ovid, and Cochrane Databases for randomized clinical trials (RCT) comparing OSS with PEG for bowel preparation before colonoscopy. The last search was performed on 22 August 2023. The primary outcome was the quality of bowel preparation. The outcomes were compared by meta-analysis and trial sequential analysis (TSA). A total of 14 RCTs with 4526 patients were included. OSS was comparable with PEG regarding adequate bowel preparation [P = 0.16, odds ratio (OR) = 1.19, 95% confidence interval (CI) [0.93, 1.51], I2 = 0%]. However, OSS showed obvious priority in excellent bowel preparation (P < 0.001, OR = 1.62, 95% CI [1.27, 2.05], I2 = 0%) and total Boston bowel preparation scale (BBPS) [P = 0.02, weighted mean difference (WMD) = 0.27, 95% CI [0.05, 0.50], I2 = 84%]. Additionally, the detection rate of polyps (P = 0.001, OR = 1.44, 95% CI [1.15, 1.80], I2 = 0%) and adenoma (P = 0.007, OR = 1.22, 95% CI [1.06, 1.42], I2 = 0%) was significantly higher in the OSS group. The two groups showed comparable incidence of adverse events except for a higher incidence of dizziness (P = 0.02, OR = 1.74, 95% CI [1.08, 2.83], I2 = 11%) was indicated in the OSS group. Moreover, OSS was associated with a higher satisfaction score (P = 0.02, WMD = 0.62, 95% CI [0.09, 1.15], I2 = 70%). In the TSA, the cumulative Z-curve crossed both the conventional boundary and trial sequential monitoring boundary and the required information size has been reached for excellent bowel preparation and total BBPS. The current data demonstrated that OSS was associated with better quality of bowel preparation. More clinical trials are still needed to confirm other outcomes.

Research on agricultural monitoring and progressive map creation in the context of ground-air coordination

Abstract By combining network RTK technology, 2D LiDAR, and UAV, this study investigates how to achieve agricultural monitoring effectively. A collaborative agricultural monitoring scheme was designed to accurately and comprehensively gather agricultural information in response to the demand for targeted sequential monitoring at specified agricultural locations. To establish a low-cost and low computational power-dependent 3D map of obstacle point clouds, a progressive 3D mapping scheme based on 2D LiDAR was designed and implemented. The effectiveness of the design was validated through simulation experiments and partly through real-world testing.

Effect of Prophylactic Tropisetron on Post-Operative Nausea and Vomiting in Patients Undergoing General Anesthesia: Systematic Review and Meta-Analysis with Trial Sequential Analysis.

This systematic review and meta-analysis of randomized controlled trials (RCTs) with trial sequential analysis (TSA) aimed to comprehensively evaluate and compare the efficacy of the prophylactic administration of tropisetron in the prevention of the incidence of post-operative nausea and vomiting (PONV) in patients undergoing surgery under general anesthesia. This study was registered with PROSPERO (CRD42024372692). RCTs comparing the efficacy of the perioperative administration of tropisetron with that of a placebo, other anti-emetic agents, or a combination of anti-emetic injections were retrieved from the databases of Ovid-MEDLINE, Ovid-EMBASE, the Cochrane Central Register of Controlled Trials, and Google Scholar. The frequency of rescue anti-emetic use (RA) and the incidence of PON, POV, and PONV (relative risk [RR]: 0.718; 95% confidence interval [CI] 0.652-0.790; I2 = 0.0, RR: 0.587; 95% CI 0.455-0.757; I2 = 63.32, RR: 0.655; 95% CI 0.532-0.806; I2 = 49.09, and RR: 0.622; 95% CI 0.552-0.700; I2 = 0.00, respectively) in the tropisetron group were lower than those in the control group; however, the incidence of complete response (CR) was higher in the tropisetron group (RR: 1.517;95% CI 1.222-1.885; I2 = 44.14). TSA showed the cumulative Z-curve exceeded both the conventional test and trial sequential monitoring boundaries for RA, PON, POV, and PONV between the tropisetron group and the control group. Thus, the prophylactic administration of tropisetron exhibited superior efficacy in the prevention of PON, POV, and PONV. Furthermore, a lower incidence of RA and a higher incidence of CR were observed with its use.

Evaluation of Immune Status of Pigs against Classical Swine Fever for Three Years after the Initiation of Vaccination in Gifu Prefecture, Japan.

In 2018, classical swine fever (CSF) reemerged in Gifu Prefecture, Japan, after 26 years of absence, and vaccination of domestic pigs using a live attenuated vaccine was initiated in 2019. Because the vaccine efficacy in piglets is influenced by the maternal antibody levels, vaccination should be administered at the optimal age by assuming the antibody level in sows. In this study, the shift in the antibody titer distribution in sows due to the initiation of vaccination to naïve herds and its influence on the vaccine-induced immunity rate in fattening pigs were investigated for 3 years. The results indicated that higher antibody titers were induced in first-generation sows after vaccine initiation because they were immunologically naïve, but the distribution of antibody titers shifted to lower levels along with their replacement with second-generation sows. The average vaccination age of fattening pigs became earlier year by year, and the vaccine-induced antibody rate was almost ≥80%. Based on the estimation of the optimal age for vaccination, it was found that vaccination at a younger age may reduce the risk of CSF infection. Taken together, the risk of CSF outbreaks can be reduced by administering vaccines at the optimal age based on the sequential monitoring of the sow's immune status.

Bayesian sequential monitoring strategies for trials of digestive cancer therapeutics

BackgroundNew therapeutics in oncology have presented challenges to existing paradigms and trial designs in all phases of drug development. As a motivating example, we considered an ongoing phase II trial planned to evaluate the combination of a MET inhibitor and an anti-PD-L1 immunotherapy to treat advanced oesogastric carcinoma. The objective of the paper was to exemplify the planning of an adaptive phase II trial with novel anti-cancer agents, including prolonged observation windows and joint sequential evaluation of efficacy and toxicity.MethodsWe considered various candidate designs and computed decision rules assuming correlations between efficacy and toxicity. Simulations were conducted to evaluate the operating characteristics of all designs.ResultsDesign approaches allowing continuous accrual, such as the time-to-event Bayesian Optimal Phase II design (TOP), showed good operating characteristics while ensuring a reduced trial duration. All designs were sensitive to the specification of the correlation between efficacy and toxicity during planning, but TOP can take that correlation into account more easily.ConclusionsWhile specifying design working hypotheses requires caution, Bayesian approaches such as the TOP design had desirable operating characteristics and allowed incorporating concomittant information, such as toxicity data from concomitant observations in another relevant patient population (e.g., defined by mutational status).

Sequential Monitoring Using the Second Generation P-Value with Type I Error Controlled by Monitoring Frequency

The Second Generation P-Value (SGPV) measures the overlap between an estimated interval and a composite hypothesis of parameter values. We develop a sequential monitoring scheme of the SGPV (SeqSGPV) to connect study design intentions with end-of-study inference anchored on scientific relevance. We build upon Freedman’s “Region of Equivalence” (ROE) in specifying scientifically meaningful hypotheses called Pre-specified Regions Indicating Scientific Merit (PRISM). We compare PRISM monitoring versus monitoring alternative ROE specifications. Error rates are controlled through the PRISM’s indifference zone around the point null and monitoring frequency strategies. Because the former is fixed due to scientific relevance, the latter is a targettable means for designing studies with desirable operating characters. An affirmation step to stopping rules improves frequency properties including the error rate, the risk of reversing conclusions under delayed outcomes, and bias.

The FIPO23 trial: First-in-human phase I/II study of XON7 in advanced solids tumors.

TPS2673 Background: XON7 is a first-in-class glyco-humanized polyclonal antibody (GH-pAb) targeting selectively multiple tumor-associated antigens. XON7 induces tumor cell apoptosis and promotes the elimination of tumor cells by immune effector cells through CDC (Complement Dependent Cytotoxicity) and ADCP (Antibody-Dependent Cell Phagocytosis). XON7 demonstrated anti-tumor efficacy in mice xenograft models with human solid tumors such as colon, prostate, lung and triple negative breast cancers. Safety pharmacology and toxicology studies in non-human primates demonstrated an acceptable safety profile for XON7. Taken together, these preclinical data support the initiation of human trials in patients with advanced solid tumors. Methods: First-in-patient, multicenter, open-label, two phases (dose-escalation (ESC) followed by dose expansion (EXP)) study of XON7 single agent, in patients with relapsed refractory, locally advanced or metastatic solid tumors without standard available treatments ( NCT06154291). Key eligibility criteria include disease progression after ≤ 4 lines of therapy; age &gt; 18 years; ECOG performance status ≤2; adequate organ functions; no known CNS involvement. All advanced or metastatic tumor types except glioblastoma, can be included during ESC. Primary endpoints: safety, tolerability and determination of MTD/RP2D. Secondary endpoints: pharmacokinetics (PK), pharmacodynamics, immunogenicity and preliminary anti-tumor activity (RECIST 1.1). AEs graded according to CTCAE 5.0. In the ESC part, XON7 is administered as 60-min intravenous infusion every 2 weeks according to an escalating schedule of doses from 1.5 to 20 mg/kg for up to 12, 28-day cycles. Dose escalation/de-escalation is based on the Bayesian Optimal Interval (BOIN) design, up to 45 pts are planned. At the recommended dose, up to 7 EXP cohorts (30 pts each) defined according to the primary tumor site are planned. Bayesian sequential monitoring will be used. Patient recruitment is planned or ongoing at 25 sites worldwide. Results: As of Feb 3rd, 2024, 3 pts are enrolled in the first dose cohort and have received XON7 at dose 1,5 mg/kg Q2W for the first 28-days cycle. Conclusions Accrual is ongoing in this first-in-human trial of XON7. Clinical trial information: NCT06154291 .

Meta-analysis: Over-the-scope clips in patients at high risk of re-bleeding following upper gastrointestinal tract bleeding.

Acute non-variceal upper gastrointestinal bleeding (UGIB) is challenging in patients at high risk of re-bleeding in whom standard endoscopic treatment (ST) has limited effectiveness. Over-the-scope clips (OTSC) have shown promise in these patients although their precise role remains uncertain. To confirm the role of OTSC in patients with UGIB at high risk of re-bleeding. We systematically searched CENTRAL, MEDLINE and Embase from January 1st, 1970 to April 24, 2024 for randomised controlled trials (RCTs) comparing OTSC and ST in acute non-variceal UGIB with high re-bleeding risk. The GRADE framework assessed evidence certainty, while trial sequential analysis (TSA) controlled random errors and evaluated conclusion validity. We analysed four RCTs (319 patients); pooled risk ratio (RR) for clinical success at initial endoscopy favoured OTSC (RR = 1.30, 95% CI = 1.08-1.56, p = 0.006, I2 = 58%, moderate certainty of evidence). TSA showed the desired sample size was 410 and the cumulative Z curve crossing the trial sequential monitoring boundary. The pooled RR for re-bleeding within 30 days favoured OTSC (RR = 0.53, 95% CI = 0.30-0.94, p = 0.03, I2 = 0%, moderate certainty of evidence). There was no significant difference in 30-day mortality, or length of hospital or ICU stay. Moderate certainty evidence supports OTSC as a superior initial treatment for acute non-variceal UGIB with high re-bleeding risk. Further large-scale studies are needed to confirm OTSCs' role by exploring other prognostic outcomes and assessing cost-effectiveness and potential complications.

Sequential monitoring for conditional quantiles of general conditional heteroscedastic time series models

AbstractIn this study, we introduce an online monitoring procedure designed to sequentially detect change points in the conditional quantiles of location‐scale time series models. This statistical process control issue holds great significance in risk management, particularly in measuring the value‐at‐risk or expected shortfall of financial assets. Our approach employs suitable detectors, including cumulative sum statistics. We then define a stopping rule and determine control limits based on asymptotic theorems to signal an anomaly. To further evaluate the proposed methods, we conduct a comprehensive empirical study analyzing various aspects of our monitoring procedures when applied to location‐scale time series models. Additionally, we perform a real data analysis using the daily returns of the Korea Composite Stock Price Index (KOSPI) and EuroStoxx 50 indices to affirm the adequacy of the proposed monitoring procedures in real‐world applications.

A coumarin derived turn ‘off–on-off’ probe for selective sequential monitoring of Al3+ and Cu2+ ions with bio-imaging application

Herein, a coumarin-based chemosensor (E)-N'-(1-(2-oxo-2H-chromen-3-yl)ethylidene) thiophene-2-carbohydrazide (ACT) has been synthesized and thoroughly characterized by using various spectroscopic techniques. The molecular structure of the probe has been confirmed by single crystal X-ray diffraction technique. Hirshfeld surface analysis has also been carried out to investigate weaker interactions in the probe molecule. ACT shows a highly selective enhanced fluorometric response towards Al3+ in ethanol among different competing cations. Upon interaction with Al3+, a new complex ACT-Al3+ is formed which displays a strong yellow fluorescence due to chelation enhanced fluorescence process (CHEF). Furthermore, in situACT-Al3+ complex is exploited for sequential recognition of Cu2+ with fluorescence turn-off via paramagnetic quenching mechanism among different competing cations. This study presents an exceptionally low detection limits of ACT for Al3+ and Cu2+ ions (2.33 × 10−11 and 1.96 × 10−11 M, respectively). Furthermore, the binding constants of ACT for Al3+ and Cu2+ were found to be 7.97 × 104 and 9.79 × 104 M−1, respectively. The binding mechanism has been verified by 1H NMR titration and DFT calculations. Sensing behaviour of ACT towards Al3+ and Cu2+ have also been exploited in the development of paper strip kit and intracellular application in 3rd instar larvae of Drosophila.

Cardiac differentiation of chimpanzee induced pluripotent stem cell lines with different subspecies backgrounds.

The comparative analysis between humans and non-human primates is an instrumental approach for elucidating the evolutional traits and disease propensity of humans. However, in primates, cross-species analyses of their developmental events have encountered constraints because of the ethical and technical limitations in available sample collection, sequential monitoring, and manipulations. In an endeavor to surmount these challenges, in recent years, induced pluripotent stem cells (iPSCs) have garnered escalating interest as an in vitro tool for cross-species analyses between humans and non-human primates. Meanwhile, compared to humans, there is less information on in vitro differentiation of non-human primate iPSCs, and their genetic diversity including subspecies may cause different eligibility to in vitro differentiation methods. Therefore, antecedent to embarking on a comparative analysis to humans, it is a prerequisite to develop the efficacious methodologies for in vitro differentiation regardless of the intraspecies genetic background in non-human primates. In this study, we executed the in vitro differentiation of cardiomyocytes from four chimpanzee iPSC lines with different subspecies and individual backgrounds. To induce cardiomyocytes from chimpanzee iPSCs, we evaluated our methodology for in vitro cardiac differentiation of human iPSCs. Eventually, with minor alterations, our cardiac differentiation method was applicable to all chimpanzee iPSC lines tested as assessed by the expression of cardiac marker genes and the beating ability. Hence, our in vitro differentiation method will advance iPSC-based research of chimpanzee cardiac development and also hold possible utility to cross-species analyses among primate species.

Robust Sequential Integrity Monitoring for Positioning Safety in GNSS/INS Integration

Robust Sequential Integrity Monitoring for Positioning Safety in GNSS/INS Integration

A comparative study of two thienopyrimidine Schiff base probes for sequential monitoring of Ga3+ and Pd2.

Two Schiff base probes (S1 and S2) were prepared and synthesized by incorporating thienopyrimidine into salicylaldehyde or 3-ethoxysalicylaldehyde individually, with the aim of detecting Ga3+ and Pd2+ sequentially. Upon chelation with Ga3+, S1 and S2 exhibited fluorescence enhancement in DMSO/H2O buffer. Both S1-Ga3+ and S2-Ga3+ were quenched by Pd2+. The limit of detection for S1 in response to Ga3+ and Pd2+ was 2.86 × 10-7 and 4.4 × 10-9M, respectively. For S2, the limit of detection for Ga3+ and Pd2+ was 4.15 × 10-8 and 3.0 × 10-9M, respectively. Furthermore, the complexation ratios of both S1 and S2 with Ga3+ and Pd2+ were determined to be 1:2 through Job's plots, ESI-MS analysis, and theoretical calculations. Two molecular logic gates were constructed, leveraging the response behaviors of S1 and S2. Moreover, the potential utility of S1 and S2 for monitoring Ga3+ and Pd2+ in domestic water was verified.

Information-based group sequential design for post-market safety monitoring of medical products using real world data.

Real world healthcare data are commonly used in post-market safety monitoring studies to address potential safety issues related to newly approved medical products. Such studies typically involve repeated evaluations of accumulating safety data with respect to pre-defined hypotheses, for which the group sequential design provides a rigorous and flexible statistical framework. A major challenge in designing a group sequential safety monitoring study is the uncertainty associated with product uptake, which makes it difficult to specify the final sample size or maximum duration of the study. To deal with this challenge, we propose an information-based group sequential design which specifies a target amount of information that would produce adequate power for detecting a clinically significant effect size. At each interim analysis, the variance estimate for the treatment effect of interest is used to compute the current information time, and a pre-specified alpha spending function is used to determine the stopping boundary. The proposed design can be applied to regression models that adjust for potential confounders and/or heterogeneous treatment exposure. Simulation results demonstrate that the proposed design performs reasonably well in realistic settings.

Circulating tumor plasma cells and peripheral blood measurable residual disease assessment in multiple myeloma patients not planned for upfront transplant.

Circulating tumor plasma cells (CTPCs) provide a noninvasive alternative for measuring tumor burden in newly diagnosed multiple myeloma (NDMM). Moreover, measurable residual disease (MRD) assessment in peripheral blood (PBMRD) can provide an ideal alternative to bone marrow MRD, which is limited by its painful nature and technical challenges. However, the clinical significance of PBMRD in NDMM still remains uncertain. Additionally, data on CTPC in NDMM patients not treated with transplant arescarce. We prospectively studied CTPC and PBMRD in 141 NDMM patients using highly sensitive multicolor flow cytometry (HS-MFC). PBMRD was monitored at the end of three cycles (PBMRD1) and six cycles (PBMRD2) of chemotherapy in patients with detectable baseline CTPC. Patients received bortezomib-based triplet therapy and were not planned for an upfront transplant. Among baseline risk factors, CTPC ≥ 0.01% was independently associated with poor progression-free survival (PFS) (hazard ratio [HR] = 2.77; p = 0.0047) and overall survival (OS) (HR = 2.9; p = 0.023) on multivariate analysis. In patients with detectable baseline CTPC, undetectable PBMRD at both subsequent time points was associated with longer PFS (HR = 0.46; p = 0.0037), whereas detectable PBMRD at any time point was associated with short OS (HR = 3.25; p = 0.004). Undetectable combined PBMRD (PBMRD1 and PBMRD2) outperformed the serum-immunofixation-based response. On multivariate analysis, detectable PBMRD at any time point was independently associated with poor PFS (HR = 2.0; p = 0.025) and OS (HR = 3.97; p = 0.013). Thus, our findings showed that CTPC and PBMRD assessment using HS-MFC provides a robust, noninvasive biomarker for NDMM patients not planned for an upfront transplant. Sequential PBMRD monitoring has great potential to improve the impact of the existing risk stratification and response assessment models.

Quantifying effect of faradaic imbalance and crossover on capacity fade of vanadium redox flow battery

In this work, we aimed to reveal two main contributors to the capacity fade of a vanadium redox flow battery (VRFB). These contributors are the oxidative imbalance caused by the hydrogen evolution reaction (HER) competing with V3+ reduction during charging, and crossover, particularly the net transfer of vanadium ions from negolyte to posolyte. To investigate this, we performed VRFB cycling under various operation conditions with sequential monitoring of the electrolytes composition. We discovered that shortly after cycling starts, the crossover makes the negolyte capacity-limiting. This leads to high polarization of the negative electrode inducing HER and increasing average oxidation state (AOS) of the electrolyte. Our examination shows that the magnitude of the oxidative imbalance correlates with that of the crossover, both being dependent on the state-of-charge (SoC). Therefore, by changing operation conditions, we can slightly influence the crossover, while dramatically affect the oxidative imbalance. We also found that the resulting magnitude of the capacity fade is a trade-off between these two side-processes. This necessitates careful optimization of the battery and electrolyte composition, along with its cycling regime. From the data obtained, we conclude that the only proper approach to achieve lower capacity fade is by ensuring that the negolyte is capacity-limiting over long-term cycling. We hope that the data on the capacity fade mechanisms presented here will facilitate development of more stable and cost-effective VRFBs.