Assessing intraspecific variation in plant volatile organic compounds (VOCs) involves pitfalls that may bias biological interpretation, particularly when several laboratories collaborate on joint projects. Comparative, inter-laboratory ring trials can inform on the reproducibility of such analyses.Tanacetum vulgare, which shows a remarkable diversity in terpenoids, forming so-called chemotypes. We performed our ring-trial with two chemotypes to examine the sources of technical variation in plant VOC measurements during pre-analytical, analytical, and post-analytical steps.In a ring trial involving five laboratories, we investigated the reproducibility of VOC collections with polydimethylsiloxane (PDMS) and analyses by thermal desorption-gas chromatography-mass spectrometry (TD-GC-MS). As model plant we used Monoclonal root cuttings were generated in one laboratory and distributed to five laboratories, in which plants were grown under laboratory-specific conditions. VOCs were collected on PDMS tubes from all plants before and after a jasmonic acid (JA) treatment. Thereafter, each laboratory (donors) sent a subset of tubes to four of the other laboratories (recipients), which performed TD-GC-MS with their own established procedures.Chemotype-specific differences in VOC profiles were detected but with an overall high variation both across donor and recipient laboratories. JA-induced changes in VOC profiles were not reproducible. Laboratory-specific growth conditions led to phenotypic variation that affected the resulting VOC profiles.Our ring trial shows that despite large efforts to standardise each VOC measurement step, the outcomes differed both qualitatively and quantitatively. Our results reveal sources of variation in plant VOC research and may help to avoid systematic errors in similar experiments.The online version contains supplementary material available at 10.1007/s11306-023-02026-6. Plants produce a tremendous diversity of specialised (secondary) metabolites that differ in concentration and composition both across and within plant species , in which multiple participating laboratories evaluate results obtained from a joint pool of samples  pre-analytical, (2) analytical and (3) post-analytical steps that may introduce technical variation in the obtained results , as a model species. This plant species displays a huge intraspecific variation of terpenoids in leaves and flowers, allowing to group individuals into distinct chemotypes  detected compounds in the recipient laboratories. Because we performed a full factorial design with two chemotypes and a JA treatment, we also tested whether the reproducibility depends on the chemotype or induction status.To assess the reproducibility of VOC analyses via PDMS coupled with TD-GC-MS, monoclonal plants of T. vulgare plants were collected from a population located in Bielefeld, Germany. Seeds were germinated in one laboratory (L5) on glass beads in a climate chamber  and the profile of the other was dominated by three terpenoids, i.e. (Z)-myroxide, santolina triene and artemisyl acetate (hereafter mixed-chemotype). The two individuals were reproduced monoclonally by propagating an adequate number of root cuttings to keep the genetic background within chemotype constant. The root cuttings were grown for another few weeks, before sending them to the participating laboratories.In January 2019, seeds of two T. vulgare plants (hereafter donor laboratories), of which four of these laboratories performed the TD-GC-MS measurements of the VOC samples received  in May 2021. The delivery also contained all other material needed for performing the experiment . PDMS tubes (5 mm length) were prepared following Kallenbach et al., (Five laboratories L1-L5) participated in the ring trial collecting and exchanging VOC samples from -L5 parti. One day before the VOC collection started, all donor laboratories determined the plant height and the number of expanded leaves as phenotypic parameters. VOC collection was performed by each donor laboratory according to a strict protocol  as follows: 4 h after the onset of the photoperiod, the youngest fully developed leaf per plant was enclosed in a 600 mL polyethylene terephthalate (PET) cup  by inserting it through a hole made in the bottom of the cup. The cup was fixed in position with a balloon stick. Plants were allowed to recover from handling stress for one day, as mechanical damage after handling can lead to higher VOC emission rates  solved in n-heptane  was applied on a filter paper (1 cm2) and allowed to evaporate for a few minutes. Then, the paper was inserted through the opening of the cup and placed onto the leaf, serving as internal standard. Next, twelve PDMS tubes were inserted per cup using cleaned forceps, ideally neither touching each other nor the leaf. To assess the volatile background, blank samples using empty cups were prepared (hereafter blank controls). A balloon stick was used to hold the empty cup above a pot containing substrate only. As for the leaf sampling, one filter paper with internal standard and twelve PDMS tubes were inserted per cup. The relative air humidity, light intensity and air temperature were measured once during the VOC collection. After six hours, the PDMS tubes were removed from the cups without touching the leaves and equally divided into six 1.5 mL glass vials, resulting in two PDMS tubes per vial. The glass vials were sealed with polytetrafluoroethylene (PTFE) tape and stored at − 20 Four hours after the onset of the photoperiod of the following day, the VOC collection was initiated. Ten µL of 100 ng µLOne day after the first VOC collection and four hours after onset of light, a herbivore damage was simulated by treating all plants with 10 mL of a 0.005% JA solution [JA diluted in double de-ionised water containing 0.1% (v:v) Triton X-100 as surfactant (Sigma-Aldrich)]. The JA solution was injected in three portions into the substrate around the stem of each plant as well as into the substrate of the control pots. In some laboratories, it was noted that the JA did not go in solution very well. One day after the JA application, again a new filter paper freshly treated with 1-bromodecane and twelve fresh PDMS tubes were inserted into each cup. For nine plant samples in L3, no sufficient 1-bromodecane was left and could thus not be added. This was also the case for twelve blank samples in L1, four blank samples in L2, and nine blank samples in L3. Environmental conditions were measured again, as described above. After six hours of sampling, PDMS tubes were removed, distributed in separate glass vials sealed with PTFE tape with two tubes per vial, and vials frozen at − 20 From each sampled plant, one vial with PDMS tubes collected before and one with tubes collected after the JA treatment as well as vials with PDMS tubes from the respective blank controls were sent to each of the recipient laboratories . After shipping without additional cooling, samples were again stored at − 20 p-cymene, α-pinene  and (Z)-hex-3-enyl-acetate  were applied directly in TD-glass tubes and measured in a concentration series .Recipient laboratories analysed the received PDMS tubes using a protocol modified after Kallenbach et al. , adjusteT. vulgare samples. Compounds were semi-quantified based on the extracted ion chromatogram (EIC) of the corresponding peak spectrum. Obtained peak areas were normalised according to the fresh weight of the respective VOC-sampled T. vulgare leaf.Recipient laboratories used their laboratory-specific infrastructure to annotate compounds from the obtained TD-GC-MS chromatograms. Compounds were annotated comparing the RI to those published by Adams  and availm function from the stats v4.2.1 package and Anova function from the car v3.1-0 package  was performed using the aovp function from the lmPerm v2.1.0 package  among recipient laboratories were calculated separately for each chemotype but also for blank samples. For this, the icc function from the irr v0.84.1 package  and samples were therefore pooled for both LMPerm and the ICC.To assess whether the number of samples in which 1T. vulgare chemotype were plotted to determine the degree of overlap between the VOC profiles measured by the different recipient laboratories  were compared between donor laboratories and recipient laboratories, respectively.Separate Venn diagrams per es Venn, . ConsidemetaMDS function of the vegan v2.6-2 package  before data transformation. The distance matrix was applied to a multivariate analysis of dispersion (betadisper) using the betadisper function in the vegan package to check whether the variability between samples differed in a laboratory-specific and chemotype-specific manner. The betadisper analysis was followed by a non-parametric multivariate analysis of variance (npMANOVA) to test for chemotype-specific variation in the VOC profiles using the adonis2 function in the vegan package. In npMANOVA with chemotype as explanatory variable, permutations were constrained to recipient laboratories nested within donor laboratories. For both betadisper analysis and npMANOVA as well as the stacked barplots, only data (relative amount in percentage) from the untreated plants were used to test for reproducibility of the PDMS approach without the confounding effect of defence induction. VOC profiles of untreated leaves were visualised using stacked barplots split by chemotype, donor laboratory and recipient laboratory, respectively. NMDS, betadisper and npMANOVA were conducted on the full dataset, whereas stacked barplots were confined to samples where at least one of the eight jointly detected compounds was found.To capture the variability of the obtained VOC profiles by the participating donor and recipient laboratories, respectively, non-parametric multidimensional scaling (NMDS) was performed, using the Kulczynski distance on square root-transformed and Wisconsin double-standardised data in the capscale function from the vegan package. To avoid collinearity of the explanatory variables, from the four plant traits measured one representative trait (plant height) was selected from a correlation matrix using Pearson correlation, as it was the only trait that correlated with the other phenotypic traits. Significance in dbRDA was assessed using permutation tests based on 9999 permutations. To test whether the changes in VOC profiles according to JA treatment are consistent, the log2-fold change (log2FC) was calculated per donor laboratory on each of the compounds jointly detected by the recipient laboratories. For each compound, the fold change (FC) was calculated as the ratio of the peak area of the JA-induced sample compared to the respective control sample for each recipient laboratory within each donor laboratory. All FC-values were then log2-transformed and median-averaged per donor laboratory for comparison. To avoid infinite numbers in the calculation of log2FC values, a constant was added that was two orders of magnitude lower than the peak area of the smallest compound detected per recipient laboratory , placing zero-value peak areas within the background noise. Variation in log2FC values was analysed based on individual log2FC values using longitudinal linear mixed-effects models with donor laboratory, VOC and their interaction as fixed effects, and recipient laboratory as well as plant individuals nested within recipient laboratory as random effect. Models were applied separately for each chemotype and log2FC values were normalised using ordered quantile normalisation. The significance of model terms was analysed with likelihood-ratio tests by comparing the original model to models reduced for each model term. Univariate data were visualised with either a point-range plot using the arithmetic average and standard deviation (i.e. for number of detected 1-bromodecane peaks per recipient laboratory), box-and whisker plots or a heatmap (i.e. the effect of JA induction). A workflow of the main statistical analyses performed in relation to the pre-analytical, analytical and post-analytical steps is provided in Supplementary Fig. S3.To assess whether the resulting VOC profile per sample was affected by early-stage phenotypic variation of plants, a distance-based redundancy analysis (dbRDA) was applied using the distance matrix from NMDS and the The number of leaves per plant, measured one day before VOC collection in untreated plants, and plant height of the two chemotypes varied significantly depending on the donor laboratory  and was not affected by chemotype . The number of samples, in which the internal standard was detected, did only marginally differ among laboratories . The VOC profile of these jointly detected VOCs was significantly affected by the plant height of individuals reared at the different donor laboratories  pointing to an impact of phenotypic variation generated during pre-analytical steps on later analytical outcomes.Based on EIC-mode analyses, the number of peaks detected per sample differed significantly among both samples sent by different donor and recipient laboratories, while it was not affected by chemotype  in the mono-chemotype was dominated in almost all recipient laboratories except L1 by β-thujone (13.6–79.9%), while the abundance varied especially for p-cymene (0–27.9%), limonene (0–40.9%) and α-pinene , particularly the light conditions were quite distinct among the donor laboratories. In addition, plants may have been exposed to different conditions during mailing to all donor laboratories. High phenotypic plasticity as a response to environmental cues has been found in various clonal plant species and is an important part of biological variation  for the content and composition of essential oils, but not for morphological traits , variation in chemical defence traits such as glucosinolates seemed to vary only little within clones but levels were expected to increase under changing environmental conditions  was found to respond to changes in light and temperature conditions even within short term   different analytical hardware, (b) variation in the settings of the TD-GC-MS measurements and (c) the experience with subsequent peak annotation.T. vulgare chemotypes, the overlap of commonly detected peaks was surprisingly low and there was a slight variation in the RIs. Moreover, quality assurance & quality control (QA/QC) measures to obtain reproducible results are not uniform across metabolomics laboratories : Analytical hardware differs in sensitivity, as found, for example, for LC–MS  a solvent cut-off of the first minutes  was applied in the TD-GC-MS measurements. This is usually applied in liquid injection to avoid overloading of the MS with solvent but not needed in solvent-free headspace sampling . Thus, at least within the laboratories, the two chemotypes could be discriminated. The high variation introduced by both donor and recipient laboratories in the number of peaks per sample and the composition of the eight common terpenoids indicates that both pre-analytical steps (mirrored in significant variation between donor laboratories) and analytical as well as post-analytical steps (mirrored in significant variation between recipient laboratories) contributed to the variation in this ring trial study. Alternative adsorbents such as polyacrylate or divinylbenzene Below is the link to the electronic supplementary material.


The State doping scandal in Russia has highlighted a major discrepancy in the fight against doping in sport: on the one hand, the signatories of the Word Anti-Doping Code  are subject to a very strict regime and incur serious sanctions, while on the other hand, States, when they massively violate the rules, do not risk very important consequences in international law. For example, high ranking officials as well as the Russian state apparatus have not been affected with a few exceptions such as the Moscow antidoping laboratory. The aim of this opinion paper is to present a reflection on the different avenues that could be envisaged to make governments more accountable, especially as work is underway on the topic. The development of a true government accountability regime would allow the system to be more balanced. For more than 20 years now, governments and the sport movement have been working together to build a robust and coherent global antidoping system. The development of the fight against doping was first initiated by the world of sport with the creation of WADA and the World Anti-Doping Code. The community of States joined this movement with the adoption, under the aegis of UNESCO, and then the entry into force in 2005 of the International Convention against Doping in Sport. This Convention is now the most widely ratified 191 States) of those administered by UNESCO  articles on internationally wrongful acts . The basThe framework text for clean sport is the International Convention against Doping in Sport, which came into force in 2005 and is administered by UNESCO which is the most ratified treaty . This trOne of the few provisions developing an obligation for States is Article 3 of the Convention, which states that “[States undertake] to adopt appropriate measures at the national and international levels consistent with the principles set forth in the Code”. However, it appears from the discussions that the wording “principles of the Code” was chosen because of the opposition of certain States to simply mentioning “the Code” . HoweverOnce a breach of the obligation is established, it must be shown that the act is attributable to the State. This means that it must be shown that officials acted on behalf of the state and violated a state commitment Again, while in a case of State-sponsored doping this does not seem difficult, certain elements will need to be verified: for example, in many States the code is incorporated simply by action of the NADO. The imputation of such a violation remained uncertain.restitutio in integrum according to Factory of Chozow Case (In international law, the preferred method of reparation is zow Case . In the zow Case .While in general international law, this method of reparation might appear sufficient, it might not be satisfactory to the antidoping community. Indeed, regarding the regime applied to the signatories of the World Anti-Doping Code, a parallelism with a sanction regime applicable to governments might be desirable. However, such measures are quite rare in international law. Indeed, international liability does not, in principle, have a punitive effect. One could, however, imagine measures such as the impossibility of playing the national anthem during competitions, of nationals of a State found guilty of violation to sit on certain committees, or of making this country ineligible to host certain competitions. This would require the articulation of several instruments, some of them new, and it does not appear that the thinking has reached that point.Hypothesis 1: an amendment to the International Convention against Doping in SportThe natural route to greater state accountability would be through a revision of the core of their clean sport commitments, the International Convention against Doping in Sport.The process of amendment, provided for in Article 33 , is not Once the amendment is adopted, States would still not be bound by it. They would have to ratify the amendment before it could be invoked against them. A State is under no obligation to ratify, nor is it required to ratify successfully. Furthermore, if an amendment was intended to develop a dispute settlement mechanism whether it is new or assigns jurisdiction to the International Court of Justice, it would be surprising if it provided for punitive sanctions for non-compliance. A priori, the only modality that could be included would be satisfaction that's to say limited to the recognition of the responsibility of the State sued. Indeed, with the number of States required to submit and adopt the amendment, only a watered-down version with a limited level of coercion could be accepted by the community of States in view of current trends in international law. Indeed, consensus or the meeting of a large number of governments is very difficult: we see this in other areas such as the WTO  or the eHypothesis 2: the implementation of an accountability system through the world anti-doping codeUNESCO”. This change was explained by the fact that States, non-signatories to the Code, could not be bound by commitments to which they had not consented.Increasing State responsibility could also be achieved through the World Anti-Doping Code. In the 2021 revision of the Code, Article 22 on government participation was changed from the imperative to the conditional. For example, article 22 (At first glance, one might think that this change is regrettable, considering that any further revision should revert to the imperative. We do not share this analysis. Indeed, an overall reading of the States’ commitments and of Article 3 of the Convention shows that the States have undertaken to respect the principles of the Code. Therefore, any change in the verb tense in Article 22 is purely cosmetic.The ability of governments to sanction through the Code should be clarified both institutionally and materially. Indeed, WADA should not have this capacity alone and should rely on an independent institution to determine whether a government has violated its obligations. Once this step of qualification of the facts is achieved, the Code could develop a diversified arsenal of sanctions. The simplest could be the impossibility for nationals of the responsible State to sit for a certain period on the various committees and bodies of WADA. One could also imagine that the Code provides for the impossibility for the country to bid for or host international competitions during a certain period. It would certainly be necessary to involve the International Olympic Committee and to develop a linkage with ongoing or already awarded competitions, but these avenues remain tangible.Compared to the UNESCO Convention, such a regime could be put in place on a revision of the Code appears to be more flexible and could ensure the implementation of a uniform regime. The Code could not, however, establish a mechanism for the settlement of inter-state disputes or give jurisdiction to the ICJ. The measures that could be developed in the Code would be of a different nature but would be complementary or would make up for rigidities in international law. Obviously, these pathways are not mutually exclusive and could be implemented concomitantly, thus increasing the robustness of the system.ad hoc arbitration mechanism, but the road to the creation of such an institution may be long and winding.Usually in international law, the responsibility of a State can be brought before a dispute settlement mechanism by a victim state. In the current state of international law, it would be possible to have recourse to the International Court of Justice, but the possibility of referring to it appears very limited because the way in which States recognize competence restricts the possibilities of access. Another solution would be to have recourse to an Under its Statute, the International Court of Justice has contentious jurisdiction and advisory jurisdiction. The contentious jurisdiction is limited to States dispute and the advisory jurisdiction is only offered to UN institutions.It would therefore be conceivable for a State to attempt to bring a case before the Court against another State in relation to a possible breach of its obligations in relation to doping in sport to obtain recognition of its responsibility. However, it is necessary for States to recognize the jurisdiction of the Court. Unfortunately, the International Convention against Doping in Sport does not contain a clause conferring jurisdiction on the Court or any other dispute resolution mechanism. In the absence of this, it is necessary to check whether the State has filed an optional declaration of compulsory jurisdiction under Article 36 para 2 of the Court's Statute  which doAnother option could be the consultative competence offered to UN bodies. In this sense, it would be quite possible for the Conference of Parties to the UNESCO International Convention to submit a request for an advisory opinion to the International Court of Justice. It does not seem to be excluded either that the Bureau of the COP could have such a competence, but this remains to be determined. The purpose of advisory opinions is to clarify a point of law and not to recognize the responsibility of a State. However, the Court is quite liberal in accepting questions put to it and it is not untrue that some of the questions put to it through advisory opinions in recent years could have been put for litigation purposes. Examples of this are the opinion on the Wall in Palestine  and the Although the possibility of recourse to the International Court of Justice seems marginal, it is—to date—the only avenue available. There may be some criticism of the competence of the Hague judges in sports law or of the length of time it takes for a case to be decided by the Court. While the question of procedural delays is certainly not wrong, the question of the competence of the judges should be put into perspective, as they are generalists in international law who work daily in many fields. The establishment of international liability or the interpretation of certain treaty principles require such skills before having disciplinary skills in sports law.ad hoc arbitration mechanism to engage the responsibility of States in doping matters was developed. This idea aimed to present a voluntary mechanism. The suggested mechanism referred to an ad hoc arbitral tribunal, but its contours remained unclear. Would this tribunal have to decide on the responsibility of a government, leaving it to WADA and UNESCO to decide the consequences, or would it have an arsenal of sanctions at its disposal?At WADA's 2022 symposium, a proposal for the creation of an ad hoc arbitration mechanism by a treaty between the countries supporting the initiative. Immediately, the question of the secretariat would arise: should an existing institution offering such services, such as the Permanent Court of Arbitration, be used or should the mechanism have its own secretariat? Whichever option is chosen, cost would be a factor to be assessed. Secondly, how many arbitrators would there be? Would they be appointed by States in the event of a dispute, or should they be chosen from a predetermined list? The modalities of the composition of this list are again to be defined. In short, there are several issues that need to be resolved before such a mechanism can be considered. However, this is not impossible. Indeed, in the context of the WTO dispute settlement crisis, some States have agreed to develop a voluntary arbitration mechanism to circumvent the Appellate Body's blockage. Another problematic element is the nature of the obligations identified by this potential mechanism. Indeed, the creation of such a mechanism would not allow, prima facie, to circumscribe and specify the obligations of States. It would therefore necessarily, if the Convention does not evolve, have to interpret the existing rules and in particular the notion of principles of the World Anti-Doping Code.If this avenue seems interesting, several questions can immediately be raised. Firstly, such a body often needs a secretariat. Therefore, should this body be created under the auspices of UNESCO within the framework of the Convention with the rigidities of the amendment procedure? Another way would be to create an ad hoc dispute resolution mechanism be developed? Can the regime be supplemented by the Code? There are many ways to go and the choice will have to be made by the community of States. If things are to move forward and quickly, it would be urgent for a group of leading governments to carry this project forward so that present and future initiatives do not remain a dead letter. In political terms, a silo response developed by either UNESCO or WADA should be avoided. The responsibility of governments will only be achieved by States working together. Perhaps the next World Conference on Anti-Doping in Sport, scheduled for 2025, would be the ideal place to initiate a collective dialogue.There seems to be a consensus today to implement the responsibility of governments when they violate their antidoping obligations in sport. The Russian doping scandal has highlighted the need to be able to mobilize State responsibility—which is not the case today—in an adequate manner. Indeed, the system cannot, on the one hand, be very strict regarding the signatories of the Code and the athletes, and on the other, lax regarding the States. However, choices will have to be made: should this be done through the Convention? Should the ICJ be used? Should an


In the United States, osteopathic manipulative treatment (OMT), is a popular complementary physical health approach for the treatment of neuromusculoskeletal disorders. However, post-OMT adverse events (AEs) are poorly defined in terms of frequency, severity, and temporal evolution. To date, no benchmark for patient safety exists. To improve understanding in this field, we set out to model the landscape of patient harm after OMT.We conducted a comprehensive search of all available primary clinical research studies reporting on the occurrence of post-OMT AEs in nonpregnant, adult outpatients treated by an osteopathic physician in the United States. The methodology of eligible studies was then reviewed to select those containing the minimum required dataset to model the post-OMT AEs. The minimum required dataset consisted of four model parameters: ‘post-OMT interval’, ‘OMT encounters with post-OMT interval assessment’, ‘AEs preceded by an OMT encounter’, and ‘AE severity.’ We used the dataset extracted from selected studies to calculate a patient safety benchmark defined as the incidence rate of AEs per 100 post-OMT interval-days.n = 161/165 (98%) of total AEs observed in the literature. The cumulative incidence of mild AEs was also significantly greater (P = 0.01) than both moderate and severe grades. The benchmark incidence rate was 1.0 AEs per 100 post-OMT interval-days.From 212 manuscripts that we identified, 118 primary clinical research studies were assessed for eligibility. A total of 23 studies met inclusion criteria for methodological review, of which 13 studies passed and were selected for modeling. Mild AEs were the most frequent, accounting for The majority of post-OMT AEs observed in the primary clinical literature were of mild severity. Modeling of the combined dataset on post-OMT AEs allowed for the derivation of a patient safety benchmark that, to date, has not been established in the field of osteopathic manipulative medicine. Additional research is needed to improve model resolution during the post-OMT period. This work conceptualized a model for identifying and grading post-OMT AEs, which should facilitate future comparisons between institutions in order to continually improve patient safety standards in the field of osteopathic manipulative medicine. Osteopathic manipulative treatment (OMT) is a complementary physical health approach that ranks fourth among the most popular options used by adults in the United States , 2. OMT In an editorial, one prominent osteopathic physician proposed that the current lack of sufficient post-OMT safety data is due to the rarity of severe AEs, which limits the feasibility of conducting much needed clinical trials that are appropriately powered . While sUnderestimation of mild AEs is further enhanced in the outpatient setting where the majority of OMT is administered , 15. HerTherefore, we recognized three barriers to progress in understanding the safety of OMT. One: while the majority share of post-OMT AEs is assumed to be mild or moderate, analysis of outcomes for this distinct therapeutic class has been diluted by the prevailing non-osteopathic manual therapy literature. Two: osteopathic physicians lack a common method to identify and grade post-OMT AEs that retains clinical utility for the assessment of pain and other forms of suffering marked by significant interindividual variability. Three: current models for conceptualizing the safety of OMT are inadequate because, to date, no benchmark has been established to compare patient outcomes between clinical institutions.To overcome these barriers, we performed a comprehensive search of the available primary clinical literature reporting on adverse patient outcomes after OMT in nonpregnant, adult outpatients treated by an osteopathic physician in the United States. We adopted a set of definitions to identify and grade post-OMT AEs. Next, we devised a set of four parameters – ‘post-OMT interval’, ‘OMT encounters with post-OMT interval assessment’, ‘AEs preceded by an OMT encounter’, and ‘AE severity’ – to model post-OMT AEs. We used data from eligible studies that passed methodological review to populate model parameters. The cumulative incidence of post-OMT AEs was calculated and modeled to derive a novel patient safety benchmark in the field of osteopathic manipulative medicine, namely the incidence rate of post-OMT AEs.A comprehensive literature search was performed as described below with the most recent search date being May 14, 2023. The authors used the available institutional medical database subscriptions, including Ovid, Clinical Key, ProQuest, LWW Health Library, Medline Ultimate, PubMed, PubMedCentral, SAGE Journals, ScienceDirect, Scopus, Springer Link, EBSCO, Oxford Academic Journals, Nature, Taylor and Francis, Wiley Online Library, and Journal of the American Medical Association, and the scholarly literature search engine Google Scholar to obtain manuscripts that were restricted behind journal paywalls. As manuscripts were identified, their respective reference lists were tracked backward in time to identify relevant manuscripts. The following unfiltered search term was entered into the PubMed database: ("adverse event" OR "adverse effect" OR "adverse events" OR "adverse effects") AND osteopathic AND (OMT OR OMM). The manuscripts resulting from this search comprised level 1 of the search strategy. Manuscripts were categorized as either primary clinical research (prospective and retrospective studies), secondary clinical research , or out of scope . The titles and abstracts of references cited by each level 1 secondary clinical research manuscript were screened to identify additional primary and secondary clinical research manuscripts that appeared to be pertinent. These manuscripts comprised level 2 of the search strategy. Level 2 manuscripts were sorted as described for level 1 and so on, eventually producing levels 3, 4, and 5. In other words, the search strategy required tracking reference lists backward in time through a chain of five referenced manuscripts. Duplicate references, identified by title, author list, and year, were discarded to avoid duplication. An attempt was made to obtain a copy of all manuscripts using the search tools described above. The authors did not contact the corresponding authors to obtain inaccessible manuscripts and, because the authors did not pay for manuscript access as the enclosed study was not funded, manuscripts that could not be accessed by the authors were labelled as ‘unable to obtain’ and were not assessed.All primary clinical research manuscripts that were identified through the search strategy were assessed to determine eligibility for inclusion in the subsequent methodological review. Inclusion criteria were as follows: study subjects age ≥ 18 years old, study subjects nonpregnant or ≥ 1 year postpartum, study subjects received OMT, OMT performed or supervised by an osteopathic physician, non-inpatient clinical setting , and study conducted in the United States. Manuscripts that failed to satisfy all six inclusion criteria were excluded.The methodologies of eligible studies were reviewed to select those containing the data required for model synthesis. We selected studies that reported data in terms of the following four parameters defined in greater detail in Table Various terms and definitions were selected to standardize the identification and grading of post-OMT AEs observed in the primary clinical literature. We adopted existing terms and definitions that were previously established in the literature. We formulated original terms and definitions as needed if no prior resource provided context. Table www.inkscape.org. Using JMP, we conducted a one-way ANOVA with blocking by study identity followed by Tukey’s post-hoc test for multiple comparisons to assess for significant differences in cumulative incidence between AE severity grades. The cutoff for statistical significance was set at P < 0.05. To calculate the benchmark incidence rate per 100 post-OMT interval-days, total modeled ‘AEs preceded by an OMT encounter’ was divided by total modeled post-OMT interval-days and multiplied by 100. Total modeled post-OMT interval-days was calculated by adding together the post-OMT interval days across all modeled OMT encounters.Studies that passed the methodological review contributed data for model synthesis. In addition to populating model parameters, study characteristics were recorded to support interpretation of results. Extraction of the parameter ‘AE severity’ was standardized as follows. If a study used the patient’s own words (POW) to indicate the occurrence of an AE, the Common Terminology Criteria for Adverse Events (CTCAE) grading scale was used to assign AE severity based on the CTCAE term and grade that best matched the affected anatomical area and POW. If a study reported the severity of AEs using an unreferenced grading system, a CTCAE grade was assigned by translating the reported severity levels in terms of the best matched CTCAE grades. Severity data was adopted as reported for studies that used the CTCAE grading system by reference. All AEs reported in the literature were categorized as AEs whether or not a causal relationship between the adverse patient outcome and preceding OMT encounter was declared and whether or not the authors declared what criteria were used to identify each AE. The parameter ‘post-OMT interval’ was converted into days for all studies. Studies with a post-OMT interval of less than 24 h were included under the post-OMT interval of one day. The OMT protocol for each study was used to construct the assessment timeline for all encounters prior to extracting the parameters ‘OMT encounters with post-OMT interval assessment’ and ‘AEs preceded by an OMT encounter.’ To calculate cumulative incidence of AEs, the parameter ‘AEs preceded by an OMT encounter’ was divided by the parameter ‘OMT encounters with interval assessment’ and the resulting decimal was multiplied by 100%. The model was graphed using JMP 15.2.1, SAS Institute Inc., Cary, NC and Inkscape 1.2.2, The Inkscape Project, As depicted in Fig. As depicted in Fig. We reviewed 23 eligible studies to determine if the respective methodologies supported data interpretation at the encounter-specific level. A total of 10 studies failed methodological review. The remaining 13 studies passed methodological review. Table n = 161/165 (98%), of total AEs outnumbered both moderate, n = 3/165 (2%) of total AEs, and severe, n = 1/165 (1%), of total AEs. No life-threatening AEs or patient deaths were observed. Figure P = 0.49), the cumulative incidence of mild AEs was found to be significantly greater (P = 0.01) than both moderate and severe AEs while the difference between moderate and severe AEs was not significant. Data for post-OMT interval days 2, 4, 5, 6, and 8 were not observed in the modeled studies. Across all severity grades and studies, n = 165 AEs were observed after n = 3,778 OMT encounters (approximately 5%). Therefore, the benchmark incidence rate of post-OMT AEs per 100 post-OMT interval-days was [165 AEs]/x[100] = 1.0 AEs per 100 post-OMT interval-days.Table  = 3/165 % of totaWe synthesized a model to standardize the evaluation of adverse patient outcomes after OMT. To do so, we addressed three barriers to progress in the field. First, we performed a search of the existing primary clinical literature to identify studies involving the administration of OMT by osteopathic physicians in the United States. That decision was made because prior efforts to determine the rate of AEs after OMT have been dominated by the body of literature reporting patient outcomes after manual therapy performed by non-osteopathic healthcare professionals – massage therapists, chiropractors, physical therapists, and non-physician osteopaths – who possess different practice rights in the United States as compared to osteopathic physicians , 5, 30. Second, we found that no common method has been used to identify and grade post-OMT AEs. We viewed this problem from the osteopathic patient’s perspective, commonly one who seeks treatment for a chronically painful musculoskeletal disorder . Two sucThird, we report a patient safety benchmark against which future trials and quality improvement studies in the field of osteopathic manipulative medicine (OMM) may be compared. The metric – AEs per 100 post-OMT interval-days – is the incidence rate modeled from the combined dataset on post-OMT AEs that we extracted after assessment of the primary clinical literature. To the best of our knowledge, the incidence rate of post-OMT AEs has never been reported. The reason may be because all prior studies conducted one post-OMT interval assessment during each post-OMT interval thereby precluding measurement of incidence rate. By modeling all studies with a standard time parameter – ‘post-OMT interval’ – we were able to derive the incidence rate of observing an AE as a function of time elapsed since prior OMT.n = 1 allopathic physician and n = 1 Canadian osteopath. While these two clinicians comprised a minority among the remaining n = 41 osteopathic physicians included in that study, the authors did not report on the number of encounters attributed to these two non-osteopathic clinicians. A third limitation is that the enclosed study is not a systematic review and was not registered with PRISMA. A fourth limitation, due to the strictness of parameters used to construct the model, is that studies reporting AEs without documentation of the corresponding post-OMT interval were excluded. A fifth limitation is that of patient recall bias due to the inclusion of studies that reported post-OMT AEs in terms of POW.This study is not without limitations. First, the lack of data for some time points, specifically post-OMT interval days 2, 4, 5, 6, and 8 as shown in Fig. During the first nine days after OMT, AEs were observed to be mild in the majority of cases. The incidence rate was benchmarked at 1.0 AEs per 100 post-OMT interval-days based on modeling data extracted from the primary clinical literature. Future research is needed to improve model resolution during the initial post-OMT period. This study should assist current research on the safety of OMT by facilitating the identification and grading of AEs after OMT.


Ultrasound shear wave elastography is a modern method that is based on measuring the shear wave velocity making it possible to determine stiffness of soft biological tissues at an arbitrary point (point elastography) or to construct a two-dimensional color image with a subsequent point measurement of stiffness  and therefore to compare the stiffness of an object with a medium or objects with each other.The aim of the study is to develop a new criterion for the comparative assessment of objects with different stiffness during shear wave elastometry: modulus of stiffness difference between object and environment.Using the original technology of building two-dimensional color elastogram, point and two-dimensional shear wave elastography were performed using linear sensors on commercial ultrasound scanners: Aixplorer , Acuson S2000 , and Verasonics acoustic system  with an open architecture to determine the stiffness values of focal inclusions and compare them with each other with the help of a new comparative elastomeric assessment criterion: modulus of stiffness difference between object and environment. First, the accuracy of the scanners under test was compared on a calibrated Elasticity QA Phantom, model 049  with a known stiffness of various inclusions and thereafter on an uncalibrated BP1901 phantom  with unknown stiffness of inclusions. The obtained values were compared to determine the influence of subjective factors on the measurement results.To assess the stiffness of the foci and compare the values with each other taking into account the rigidity of the environment, it is proposed to use a new criterion for the comparative assessment — the modulus of stiffness difference between focus and environment, which quantitatively characterizes the difference between these values. According to this criterion, all three ultrasound scanners have been established to show high and comparable accuracy in determining the stiffness of inclusions within the homogeneous medium in the experiments on phantoms. Two-dimensional shear wave elastography has revealed the effect of the control volume size and the correctness of the color scale setting, especially in the heterogeneous objects, on the results of elastometry. Methodological techniques to reduce the influence of subjective factors have also been proposed.The study has showed the possibility of using the modulus of stiffness difference between object and environment as a new criterion for comparative assessment of objects in shear wave elastometry taking into account stiffness of the environment. To reduce operator-dependence, it is necessary to take into consideration both the way of realizing elastometry  and a number of other methodological factors. Ultrasound shear wave elastography is a modern technique which allows one to quantitatively assess stiffness of soft biological tissues and to build their color image on the basis of measuring shear wave velocity for subsequent conversion to Young’s modulus , 2.There are two ways of technical implementation of this method: point shear wave elastography and two-dimensional shear wave elastography. The point elastography makes it possible to measure only quantitatively the stiffness of the tissue in the selected local area, i.e. to perform elastometry. Two-dimensional elastography provides both planar visualization of the zone of interest in various shades of the color range corresponding to the stiffness of the scanned tissues and their subsequent quantitative assessment in any area of the obtained color elastogram , 4.Two-dimensional image is achieved by creating several focused ultrasound, the so-called pushing pulses, sequentially generated at different depths with a small time delay. The wave fronts of these pulses form a widening cone (Mach cone) propagating in the tissues with the speed proportional to their stiffness; this cone may be traced using planar ultrasound waves with a high frame frequency .Since there exists a diversity of ultrasound scanners implementing one or both elastographic techniques, there arises the question regarding accuracy and reproducibility of measurement results . In thisThe reproducibility of the stiffness measurements is well evaluated on phantoms simulating soft biological tissue both with inclusions having a known Young’s modulus  and with unknown stiffness of inclusions  , 8. The The aim of the study is to develop a new criterion for the comparative assessment of objects with different stiffness during shear wave elastometry: a modulus of stiffness difference between object and environment.the first stage of the investigation, there was performed a point and two-dimensional shear wave elastography of the calibrated polymer Elasticity QA Phantom, model 049  with homogeneous cylindrical inclusions of different diameters and stiffness degree located in the uniform matrix  with various known values of Young’s modulus.The calibrated phantoms were fabricated from the Zerdine special polymer with the acoustic properties maximally approximated to the averaged physical parameters for human soft tissues: ultrasound wave propagation velocity was 1540±10 m/s, attenuation coefficient was 0.50±0.05 dB/cm.МHz, density — 1.04 g/cmStiffness was measured in the cylinders of the greatest diameter (20 mm), which allowed us to avoid inclusion into the calculation zone of shear wave propagation the cylinders with a small diameter (>10 mm) . The conthe second stage of the investigation, BP1901 uncalibrated elastographic phantom  was used; it represented a silicon model of the breast with rounded inclusions varying in echogenicity, stiffness, and degree of homogeneity randomly located in the acoustically uniform matrix. Stiffness of the inclusions was unknown and not indicated in the documentation. At first, localization, size, echogenicity, and uniformity of inclusions were evaluated using grayscale scanning. Thereafter, the quantitative values of their stiffness were determined by means of point and two-dimensional shear wave elastography.At An ultrasound investigation was carried out on three scanners with shear wave elastography technology: Aixplorer  with SL15-4 linear sensor (4–15 МHz), Acuson S2000  with 9L4 linear sensor (4–9 МHz), and open-architecture Verasonics acoustic system  with L4-7 linear sensor and a specified operating frequency of 5 MHz. Aixplorer and Verasonics scanners are designed to perform point and two-dimensional shear wave elastography, while Acuson S2000 is for the point elastography only. While measuring the velocity by two-dimensional color elastogram on Verasonics and Aixplorer scanners, it was possible to change the diameter of the control volume from 1 to 10 mm, which influenced the quantitative values in the objects with non-uniform stiffness.The Verasonics system is an ultrasound device designed to study elastic properties of various objects, to optimize technical processing of acoustic signals, and to develop the most informative setting modes for scanners. The chief advantage of the device is its openness, i.e. the possibility to change the parameters of the generated acoustic waves over a wide range including the application of shear wave elastography.Construction of two-dimensional color elastogram is based on the excitation of several consecutive sources of shear wave propagation in the medium. For this purpose, a number of focused pulses creating shear waves are generated at different depths along the ultrasound beam. Five such pulses were used on the Verasonics. The originated shear waves interact with each other forming two quasi-planar waves propagating in different directions. The quasi-planar wave has a greater amplitude than an ordinary shear wave and therefore can travel sufficient distances and penetrate different objects in the medium, while its shape allows a uniform distribution over the entire depth of the examined area.Once quasi-planar shear waves are generated, the process of registration of their propagation in the medium is carried out. For this purpose, a small sequence of imaging pulses is activated after each series of focused pulses. Their basic series is initiated already after the formation of quasi-planar waves and further propagation of these waves is registered. During data processing, displacement of the points in different coordinates of the examined area is calculated using correlation of one of the imaging pulses with the reference pulse. The wave delay caused by the medium displacement is determined by the shift of the correlation curve peak relative to zero. Calculation of the displacement allows one to visualize formation and propagation of the quasi-planar shear waves .As a result, a map of elasticity of the examined area is obtained in the form of a matrix 30×30 mm or 101×101 points in size. Frame-by-frame recording of imaging pulses registering the propagation of the quasi-planar shear wave is used for its formation. At the time of its arrival, a clearly expressed maximum of particle displacement is fixed at each point of the medium. Comparing pixel coordinates along the axis parallel to the axis of quasi-planar shear wave propagation with the time of the wave arrival to the given point and knowing the same parameters for the neighboring pixel, it is possible to calculate the velocity of the wave on the given area. Eventually, the MatLab-based original program was used to create a map of Young’s modulus distribution depending on the object stiffness in the area under study .Two-dimensional elastography technique works on the Aixplorer expert system according to the same principle. However, only the final result in the form of the ready color elasticity map is displayed for the user, with its superposition on a grayscale image of the examined area. The process of shear wave propagation itself in the medium is not visualized in this device.Since the Acuson S2000 scanner provides only point shear wave elastography and the Aixptlorer scanner performs point measurements using two-dimensional color elastogram, a comparative assessment of accuracy for both techniques was first done on the calibrated phantom to exclude the effect of technological causes on the results of measurements. We compared both numeric values of stiffness for these scanners with each other and with the data from the Verasonics scanner equipped with both elastometric options.In order to assess the focus stiffness taking into account stiffness of the environment, we propose to use not only absolute values but also to introduce a relative quantitative indicator — a modulus of stiffness difference between focus and environment. It is more preferential to use the difference of absolute values than their ratio from the standpoint of the measurement error theory, and application of the modulus allows one to assess only quantitative difference between the stiffness of the focus and the medium. In this connection, all objects may be divided by the relative stiffness into comparable with the medium, differing from it slightly, moderately, and significantly. The modulus of stiffness differencecharacterizes quantitatively the distinction between an object and a medium and reduces a well-known apparatus dependence in the evaluation of the shear wave elastometry results on the scanners from various manufactures since it is the difference of quantitative values taken into consideration during stiffness assessment, not the absolute figure.C) without automatic conversion to the Young’s modulus (E), it was done manually using a known formula Е=3ρС2, where the medium density ρ was taken equal to 1 g/cm3.Values of stiffness for the background and cylindrical inclusions, as well as moduli of their difference obtained by point shear wave elastography on the Verasonics and Acuson S2000 scanners, are presented in The results obtained are in line with those of previous studies on the calibrated spherical phantom  and showAt the second stage of the investigation, an uncalibrated phantom was used, in which 11 objects with various acoustic properties were identified, of which 3 inclusions had a heterogeneous, mainly hyperechogenic, structure, the rest differed in echogenicity but were homogeneous .Stiffness was measured for all inclusions . Since tWhile measuring the stiffness of unechogenic cyst-like inclusions using point elastography, no results were obtained, but measurements performed by two-dimensional color map elastography provided regularly quantitative values. It is difficult to judge reliably the reason for such discrepancy between the methodologies: it is most likely that the device registers low-amplitude noise signals, which are of no diagnostic value .High values of the modulus of stiffness difference characterize considerable distinction from the stiffness of the object and environment, which evidences explicitly the presence of a focus . When thBut there is another question: what is permissible non-uniformity of the biological medium, for example, the cyrrotically changed liver parenchyma, to show the ultimate modulus of stiffness difference which, when exceeded, will denote the area of non-uniformity as a focal formation? The answer requires a further in-depth study.It has been established that during elastometry using two-dimensional elastograms, the size of the control volume strongly influences local values of stiffness of hyperechogenic heterogeneous inclusions, as well as their distinction from the environment. So, if its diameter was 1 mm, the average values of local stiffness in different inclusion areas on the Aixplorer scanner had significant variations from 111 to 297 kPa, whereas at larger diameters (up to 4 mm), the spread in values reduced considerably, and at 7 mm it remained reproducible within the measurement error. In homogeneous inclusions, such a marked effect of the control volume size on the difference of the local stiffness values from the average value for the entire inclusion has not been noted.The correct choice of the control volume is especially important when small objects of 10–12 mm in diameter are examined. In this case, there exists the likelihood of the total measurement of the shear wave velocity in the object and beyond it, which may result in the distortion of the numerical values, especially if the difference of the absolute stiffness values between the object and environment is considerable . PerformUpon the whole, values of stiffness for the background and inclusions obtained on different scanners are comparable, except for the Aixplorer, whose data were systematically higher than the results of investigation on two other devices. Comparing the stiffness measurements performed on the Verasonics using point elastometry and two-dimensional color elastometry, a certain difference of the results was noted in the echogenically homogeneous inclusions expressed in smaller values of stiffness for point elastometry. Since the process of determining the shear wave velocity in the Verasonics is technically the same, operator-dependent measurements may be considered the reason for this difference. The influence of the subjective factor on the elastometric accuracy is now being studied and discussed in the world literature, and the results of these investigations are not so unambiguous , 11.During point elasometry, the physician determines the velocity of the shear wave randomly in various areas of the grayscale image of the object without having any color image of its stiffness. When measuring the local stiffness using a ready two-dimensional color elastogram, the physician immediately sees in color the stiffness differences of the object, which can influence the choice of the areas for measurement and final values. The argument in favor of this may be the fact that the results of measuring the uniform objects on the Verasonics using point elastometry appeared to be closer to the data obtained on the Acuson S2000, while a separately conducted study using two-dimensional color elastogram showed the results closer to those obtained on the Aixplorer. In the non-uniform inclusions, the results seemed to be more comparable with each other due to a greater number of measurements in the areas with various echogenicity, which resulted in averaging of the results of both methods with leveling of the difference between them.In two-dimensional elastography of a non-uniform focus or medium, an important factor of the measuring correctness and subsequent comparison is the correct adjustment of the color stiffness scale to the greatest value (in kPa or m/s) to minimize the subjective factor in the selection of the zones for point elastometry. This correctly assigned value of the stiffness scale must exceed insignificantly maximal values for the given medium or object obtained during initial experimental measurements and should be displayed in the most intensive red color. This gives an extremely wide color range of stiffness imaging and reduces the subjective factor in selecting the areas for point elastometry. If the greatest value on the scale is set too low, the areas with the smaller real stiffness value will be colored in intensive red, creating an illusion of a uniform, very stiff focus, leading to the incorrect choice of the zone for point elastometry .The results of the conducted experimental study allowed us to propose a new criterion for comparative elastomeric assessment — the modulus of stiffness difference between object and medium. The methodological aspects of its application have been developed. In order to reduce operator-dependence, it is suggested taking into account the way of elastometry implementation (point or two-dimension color elastography), as well as other factors: control window size, adjustment of the stiffness scale, the number of measurements, their distribution in the object.


Aesthetic Surgery Journal and wrote my first editorial, “Turning the Page.”1 In that first editorial I made the following observations:Journal is well positioned to stay at the forefront of clinical, scientific, and technological advances and to bring you the information you need to stay ahead of the curve. That goal, along with our commitment to patient safety, is our most important mission.”“It is inevitable that changes in healthcare—and perhaps more ups and downs in the economy—will challenge and impact all of us in the years ahead. Whatever changes come, however, we can be certain that cosmetic surgery and medicine will continue to progress at an almost overwhelmingly rapid pace. It is also clear that an increasing number of physicians and nonphysicians will enter the field. The It was an honor, with much excitement and anticipation, that on January 1, 2009 I assumed the editorship of Yes, much has changed over the past 15 years, but as I turn another page, those words are as true now as they were then.Journal are under our own control, but those pale when compared with changes we cannot control. The world around us is continually changing, and the economy remains consistent only in its cyclical ups and downs, which directly affect aesthetic surgery spending. Despite the economic cycles, interest in aesthetic surgery and cosmetic medicine continues at an impressive pace. Innovations in treatment and the development of new drugs and devices are keeping pace with the demand. Our commitment to be at the forefront of these developments, bringing evidence-based information to our readers and promoting the best interests of patient safety, remains the core mission of the Journal and that is well within our control. Decisions on what we publish, how often we publish, who serves on our editorial board, and who serves as a reviewer are all the responsibility of the editorial team.Some changes that impact the Journal's benefit now and beyond 2023.The economy, the changing landscape of scholarly publishing, the predicted end of printed journals, the continued growth of online-only publishing, the rise of open-access journals, and most recently the disruptive forces of artificial intelligence are all beyond our control, but we have the will and the ability to adapt to and accept the changes affecting us today by embracing them for the ASJ, its Impact Factor, or the rising circulation and international reach over the last 15 years. The credit for any success we have enjoyed goes to the team with whom I have been fortunate enough to work. The disappointments, which fortunately were very few, are my responsibility.My intent in this final editorial as editor-in-chief is not to chronicle the success and continued rise of I have always maintained the philosophy that the hallmark of any scholarly publication is to present divergent views and to promote debate and discussion in a civil and respectful manner. Central to our belief has always been that regardless of our success and international ranking, complacency and arrogance would remain alien to us.ASJ; Laura Simson, managing editor of ASJ Open Forum; and Anastasia Cyzewski, assistant managing editor of ASJ. To this team and the others who served along the way, I offer my sincere thanks and appreciation for your tireless commitment, enthusiasm, and contributions.It remains for me to express my gratitude to those who have made my tenure a joy and those who shared my “labor of love” commitment to a “24/7, 8 days a week” work ethic. In January 2009, the team consisted of Melissa Berbusse Schmidt, who served as executive editor, and me. As we grew, Hunter Alexander joined the team, serving as managing editor for many years. Next, Phaedra Cress was hired as executive editor, now serving as executive publisher, journals; followed by Kyleigh Vrettos, editorial associate; Abby Pugh, managing editor of During my tenure, I have had the privilege of working with 15 presidents and boards of directors of The Aesthetic Society. The boards allowed me full editorial freedom. Both the journals and I benefited from their support and that of the Society staff, led by Executive Director Sue Dykema, and Bob Stanton before her. It has been a pleasure to work with our section editors, editorial board, reviewers, and authors. Thank you. I have learned much from all of you.ASJ's associate editor and ASJ Open Forum's editor-in-chief, Dr Jeffrey Kenkel, who has been by my side for 15 years. He is a scholar, a researcher, a leader, a contributor, an outstanding surgeon, and most of all, an accomplished educator. Given his many qualifications, I have no doubt that the Journal is in the best of hands and that it will continue to enjoy many successes in the years to come.The page turns to my good friend, 2-6 That, I shall miss. It was an honor back in 2009 to be entrusted with the editorship of Aesthetic Surgery Journal, and I am honored to have served the Journal, The Aesthetic Society, our specialty, and our readers.As editor I had the privilege and pleasure of writing editorials on a regular basis.Journal is coming to an end, but this is not a retirement. I plan to continue the full practice of aesthetic surgery, and to continue to contribute to the specialty I love—and yes, I will be watching ASJ from afar.My term with the


Correction to: Journal of Molecular Medicine (2023) 101:813–828 https://doi.org/10.1007/s00109-023-02325-4Conflict of Interest Ariel E. Feldstein is currently (since July 2022) an employee and stockholder of Novo Nordisk.


Atypical hemolytic uremic syndrome (aHUS) is a rare cause of end-stage kidney disease and associated with poor outcomes after kidney transplantation from early disease recurrence. Prophylactic eculizumab treatment at the time of transplantation is used in selected patients with aHUS. We report a retrospective case note review describing transplant outcomes in patients with aHUS transplanted between 1978 and 2017, including those patients treated with eculizumab.The National Renal Complement Therapeutics Centre database identified 118 kidney transplants in 86 recipients who had a confirmed diagnosis of aHUS. Thirty-eight kidney transplants were performed in 38 recipients who received prophylactic eculizumab. The cohort not treated with eculizumab comprised 80 transplants in 60 recipients and was refined to produce a comparable cohort of 33 transplants in 32 medium and high-risk recipients implanted since 2002. Complement pathway genetic screening was performed. Graft survival was censored for graft function at last follow-up or patient death. Graft survival without eculizumab treatment is described by complement defect status and by Kidney Disease: Improving Global Outcomes risk stratification.P = 0.006) in medium and high-risk recipients with 1-y survival of 97% versus 64% in untreated patients. Our data supports the risk stratification advised by Kidney Disease: Improving Global Outcomes.Prophylactic eculizumab treatment improved renal allograft survival (Prophylactic eculizumab treatment dramatically improves graft survival making transplantation a viable therapeutic option in aHUS.  HUS is classified according to its etiology: Shiga-toxin producing Escherichia coli (accounting for 90% of cases), secondary HUS when there is an identifiable cause, and atypical HUS (aHUS).2 aHUS encompasses complement and noncomplement mediated causes.3Hemolytic uremic syndrome (HUS) is a type of thrombotic microangiopathy (TMA) and describes a group of rare, life-threatening diseases characteristically presenting with microangiopathic hemolytic anemia, acute kidney injury, and thrombocytopenia coupled with histological evidence of endothelial injury.4 and with the effective use of complement inhibition as treatment.6 Approximately, 60% to 70% of patients with aHUS have inherited or acquired dysregulation of the complement alternative pathway.3 Loss of function genetic variants are identified in genes encoding complement regulatory proteins; complement factor H (CFH), complement factor I (CFI), membrane cofactor protein (CD46), and gain of function variants in genes encoding activating proteins; C3 and complement factor B (CFB).7 For people with a disease-associated genetic variant, clinical manifestations usually develop when their genetic susceptibility is challenged by environmental triggers.The essential role of the complement alternative pathway in the pathogenesis of aHUS is well established; through genomic studies3 Until recently, treatment relied upon plasma exchange and supportive therapies, with a mortality risk in the acute phase of 10% to15% and approximately 50% of patients progressing to end-stage kidney disease (ESKD) with their first episode.9 Despite the high rate of ESKD, these patients were previously precluded from renal transplantation by the high risk of recurrent disease, the exception being for a dual liver-kidney transplant in those patients with pathogenic variants in proteins produced in the liver10 or for patients with pathogenic variants in CD46. Disease recurrence predominantly occurred in the first year and resulted in graft loss for up to 90%.12aHUS has an incidence of 0.5 per million.15 In native kidneys, an advantage has been demonstrated for early treatment.13 As most posttransplant aHUS recurrence occurs within the first year,11 prophylactic eculizumab therapy initiated at transplantation has been proposed to prevent disease recurrence and associated graft injury. Cause of complement dysregulation and previous transplant history was proposed to guide recurrence risk stratification.16 In 2016, Kidney Disease: Improving Global Outcomes (KDIGO) advised prophylactic therapy for transplant recipients considered at medium or high risk of disease recurrence.3 Data from France17 support this strategy for improving transplant outcomes in aHUS.Eculizumab, a monoclonal antibody targeting C5, blocks complement terminal pathway activity. It has revolutionized renal outcomes in aHUS for disease in both native and transplanted kidneys.18 We conducted a retrospective study of renal transplant recipients with a known diagnosis of aHUS, to assess the efficacy of prophylactic eculizumab treatment compared with patients with similar defects in complement regulation who were transplanted in the pre-eculizumab era.In the UK, patients with suspected aHUS are referred to a single-specialist center for further investigation and access to eculizumab.http://www.atypicalhus.co.uk) performs genetic screening for patients with complement mediated kidney diseases. Patients with ESKD attributed to aHUS were identified from the National Renal Complement Therapeutics Centre cohort through the combination of clinical features, renal histology, and exclusion of other causes of TMA . Screening for complement pathway abnormalities was undertaken as detailed below.The National Renal Complement Therapeutics Centre, Newcastle upon Tyne, UK  was recommended before starting eculizumab22 and prophylactic antibiotics were advised for the duration of eculizumab therapy.22Patients on eculizumab are at increased risk of CD46 due to their recognized low risk of disease recurrence.3 Given the advances in immunosuppression over the period of these transplants (1978-2016) and improved graft survival,23 we assessed outcome in transplants stratified by year of transplantation. DCD kidney transplant survival has improved most dramatically over the years with NHSBT data giving 5-y graft survival of 73% for those implanted in 1999–2001,24 compared with 86% for grafts implanted in 2002–2004 or later.26 For this reason, transplants implanted before 2002 were compared with those from 2002 onward. The lower survival in the pre-2002 transplants was consistent with an era effect . We therefore included only transplants received since 2002 in the control cohort to be compared with prophylactic treatment.As the prophylactic treatment cohort consisted only of recipients at medium or high risk of disease recurrence, a subgroup of comparable recipients in the untreated cohort was generated. This excluded recipients with rare genetic variants in C3, CFB, CFH, CFI, or CD46 was detected. Variants of uncertain significance (VUSs) in any of these genes were grouped together. When assigning transplants in factor H autoantibody (anti-FH) positive recipients to an analysis group, those with isolated anti-FH autoantibodies or concomitant VUS were assigned to the anti-FH group, whereas those with pathogenic variants went by the affected gene.To assess the impact of recipient complement defects on graft outcome, transplants in the cohort not treated with eculizumab were categorized firstly by the affected gene if a pathogenic variant in All transplants were performed before August 4, 2017 to allow 36 mo follow-up.Table S2, SDC, http://links.lww.com/TP/C577. Graft survival was reported at patient death with a functioning graft or last follow-up.Available medical notes were reviewed for all groups. Transplant recipient sex, age at transplantation, year of transplantation, graft outcome, and cause of death with functioning graft were collected. Previous kidney transplant history was collected when available. Further data points collected when available for control and prophylactic treatment cohorts are detailed in CFH, CFI, CFB, C3, and CD46 was undertaken using Sanger sequencing, as previously described.31 Screening for chromosomal rearrangements affecting CFH, CFHR1, CFHR2, CFHR3, CFHR4, CFHR5, CFI, and CD46 was undertaken using multiplex ligation-dependent probe amplification, as previously described.33 To assess for genetic abnormalities in noncomplement genes associated with aHUS including DGKE,34MMACHC, VTN, PLG, THBD, and IFN235 Sanger sequencing was performed  in selected patients.Variant screening of 36 with refinement developed by Sequence Variant Interpretation Working Group.37Rare genetic variants were evaluated using Alamut Visual 2.10 (2017 Interactive Biosoftware). Variants were classified in 2019 according to American College of Medical Genetics and Genomics guidelines38 The role of anti-FH autoantibodies in aHUS was recognized in 200539 so testing was not performed in every case.The consensus ELISA assay was performed to detect anti-FH autoantibodies, as previously described.Renal graft survival, analyzed with Kaplan-Meier curves, was censored for patient death with a functioning graft and for functioning graft at last follow. Log-rank test assessed the difference between survival of 2 groups.Group characteristics were compared using T-test for continuous variables (age) and Fisher exact test or Chi-squared tests for categorical variables as determined by expected counts. Bonferroni adjustment was used for multiple pairwise comparisons. Mann-Whitney U test compared median time with presentation, follow-up, and year of transplantation.http://www.rstudio.com/. P < 0.05 was considered statistically significant.Analysis was performed using Rstudio Team (2021). RStudio: Integrated Development Environment for R. R Studio, PBC, Boston, MA URL, This study was exempt from NHS Research Ethics Review.26To assess the impact of prophylactic eculizumab treatment we compared graft survival of transplants performed with eculizumab treatment with transplants performed after 2002 in recipients at medium and high risk of recurrence who had not received eculizumab (control cohort). Grafts implanted between 2002 and 2017 were considered comparable as there has been little change in kidney graft survival in the UK during this period.P = 0.064). Thirteen of the 14 in the prophylactic treatment group that were being retransplanted had experienced previous graft loss from posttransplant TMA .Recipient demographics, complement defect status, and transplant details are given in Table CFH were the most common, in keeping with the literature and included point mutations, exon deletions, exon duplications, premature stop codons, and chromosomal rearrangements.40 Recipients classified as high risk of aHUS recurrence based on genetic screening results, autoantibody testing, and previous transplant history,3 predominated in both groups .Complement defects were similarly distributed within each group Table . PathogeSupplemental Data Sets, SDC, http://links.lww.com/TP/C576).The prophylactic treatment cohort all had a clinical diagnosis of aHUS as cause for acute kidney injury and ultimately native ESKD. Twenty-five had a native kidney biopsy showing TMA. In the control cohort, 17 had an available kidney biopsy result of which 14 showed TMA  and were in recipients with pathological variants in CFH. C4d staining was negative in both cases. In 1 (#47) TMA was shown 4 d posttransplant with evidence of microangiopathic hemolytic anemia despite absent terminal pathway activity. She received plasma exchange, repeat eculizumab dosing and the graft continues to function. In the other (#118), initial transplant biopsy <3 mo posttransplant showed acute T cell mediated rejection but with endothelial swelling and loss of fenestrations on electron microscopy, consistent with TMA (no donor-specific antibodies detected). Subsequent biopsies did not demonstrate TMA until graft loss, 6-y posttransplant, when chronic TMA was identified. As there were concerns of incomplete complement blockade 6 mo posttransplant, the fortnightly dose was increased to 1500 mg, after which complement activity was fully blocked.In the control cohort, transplant kidney biopsy data were available for 17. Fourteen showed TMA. In the prophylactic eculizumab group, 18 kidney transplants were biopsied and 11 showed rejection. Two showed TMA (2 (n = 21). In the 3 children (aged 9-11 y), mean (SE) creatinine at 1 y was 0.56 (0.29) mg/dL and eGFR was 126.8 (29.2) ml/min/1.73 m2.In the prophylactic treatment cohort, 27 had immediate graft function and the remaining 5 with available data had delayed graft function. In adults with available data mean (SE) 1-y creatinine was 1.71 (0.25) mg/dL (n = 28) and eGFR was 60.0 (5.3) ml/min/1.73 mP < 0.001). Death-censored graft survival was significantly better with prophylactic eculizumab treatment compared with controls . In the control cohort 3-mo, 1-, and 3-y cumulative death-censored graft survival was 79%, 64%, and 61%, respectively. In those treated with prophylactic eculizumab, graft survival at the same time points was 100%, 97%, and 89% respectively. Fewer transplants were lost from posttransplant TMA in the treatment cohort compared with the control cohort .Graft outcome is detailed in Table Neisseria meningitidis B bacteremia (#300). The recipient had not received B serotype vaccination or antibiotics for meningococcal prophylaxis. This was the only case of meningococcal disease recorded.One graft treated with prophylactic eculizumab failed following SDC, http://links.lww.com/TP/C576). There were no other infection-related deaths. There were no deaths with a functioning transplant in the control cohort (P = 0.057).In the prophylactic eculizumab treatment cohort, 5 recipients died with a functioning graft, in 4 this was over 2 y posttransplant. One death was attributed to multiorgan failure associated with disseminated Candida and Herpes simplex virus infections . In a recipient with no variant (#575), eculizumab was withdrawn at 6 mo and the graft failed from chronic rejection after 5 y. The other had a CFHR1/CFH hybrid (#176) and stopped after 8 mo due to declining function without evidence of aHUS recurrence. This graft failed at 15 mo with no evidence of TMA on biopsy .Eculizumab continued for the duration of renal graft function in all but 4 transplant recipients treated with prophylactic eculizumab and 2 of these continue to function . The majority of recipients were female (60%). Mean (SD) age at transplantation was 25.6 (±13.6) y. Eighteen children were included.Eighty kidney transplants in 60 recipients with aHUS were transplanted between 1978 and 2016 and not treated with eculizumab . The distribution of complement defects is show in Table S4, SDC, http://links.lww.com/TP/C577.Given the method of identification Figure , all recTable S4, SDC, http://links.lww.com/TP/C577).Median (range) time to last follow-up was 9 y (1.8 - 28 y). No recipients died with a functioning graft during the follow-up period and 61 (76%) grafts failed by last follow-up. Graft survival by complement defect is shown in Figure CFH had high levels of early graft loss with cumulative 1- and 3-y graft survival of 42% and 35%, respectively. In those with VUS, cumulative 1- and 3-y graft survivals were 58% and 50%, respectively. Of the 4 transplants in recipients with pathological variants in CD46, 1 graft failed 10-mo posttransplant from hypertensive donor disease. The diagnosis of donor-derived hypertensive disease in this case is based on review of 3 transplant biopsies carried out between 8 d and 3 mo posttransplantation. Arteriolar hyalinosis with relative glomerular preservation persisted from the initial biopsy and the kidney never functioned well.Recipients with pathological variants in 3Graft survival Figure B was ass41 Transplantation has therefore become a viable and successful option for patients with aHUS reaching ESKD. These findings are in agreement with those reported by Zuber et al,17 thus providing further evidence for the current KDIGO approach to transplant care in aHUS.3The UK experience strongly supports the use of prophylactic eculizumab treatment at the time of transplantation to improve graft survival in patients with aHUS at medium or high risk of disease recurrence. By preventing aHUS recurrence, eculizumab treatment improved graft survival from 61% to 89% at 3 y, in line with UK outcomes for all causes of ESKD. For the decade 2001-2011, 3-y graft survival for UK adult kidney transplant recipients was 89.5% to 93.9%, depending on donor type and our prophylactic treatment cohort included predominantly deceased donor kidneys.P = 0.057). It is difficult to make a comparison between these death rates given the high rate of early graft failure in the control cohort and that data on patient survival after graft failure were not collected. At 2 y posttransplant, only 61% of grafts in the control cohort continued to function compared with 92% in the treatment cohort and 80% of recipients that died with a functioning graft did so after this time point. As such, the lower death rate with a functioning graft in the control cohort could reflect the low rate of graft survival rather than reduced patient mortality.It was noted that 5 recipients (13%) in the treatment cohort died with a functioning graft compared with none in the control cohort  and will ultimately determine whether eculizumab withdrawal is safe, in whom it should be attempted, and with what surveillance. Further work is needed to stratify risk of disease recurrence posttransplantation to inform decisions regarding treatment duration and more detailed variant analysis is likely to contribute to this.Given the significant cost and infection risk of eculizumab treatment, the optimal duration of treatment is being investigated. STOPECU (NCT02574403) demonstrated a 23% relapse rate in native kidney disease after eculizumab withdrawal.17 When other precipitants of endothelial injury can be minimized, a reactive approach could be considered. Ultimately, the numbers studied remain low so this area would benefit from more targeted research.In summary, our study demonstrates prophylactic eculizumab treatment significantly improves kidney transplant survival in patients with aHUS with at least medium risk of disease recurrence, making it a viable and superior strategy for renal replacement therapy in these patients. We advocate prophylactic eculizumab treatment before transplantation, rather than reactive treatment posttransplant, as the better option based on currently available data.UK aHUS Transplant Consortium: Aimun Ahmed, Damien Ashby, Atif Awan, Richard Baker, Sunil Bhandari, Coralie Bingham, Girish Bommayya, David Border, Cormac Breen, Henry Brown, Alison Brown, Paul Carmichael, Paramit Chowdhury, Simon Curran, Rosie Donne, Chris Dudley, Tina Dutt, Kevin Eardley, Elinor Eblamo-Abad, Daniel Gale, Sian Griffin, John Harty, Peter Hewins, Richard Holt, Victoria Ingham, Hannah Kilbride, Ed Kingdon, Robert Lewis, Nicholas Mansfield, Stephen D Marks, Phil Mason, Paul Mead, Muir Morton, Thalakunte Muniraju, Pramod Nagaraja, Neal Padmanabhan, Nicholas Plant, Tara Raftery, Peter Rowe, Alan Salama, Packiam Shenbagaraman, Alison Taylor, Kerry Tomlinson, Nick Torpey, Mark Uniacke, David Walbaum, Michelle Webb, Matt Williams, Alastair Woodman, Sapna Shah, Imran SaifThis work uses data provided by patients and collected by the NHS as part of their care and support and would not have been possible without access to this data. The NIHR recognizes and values the role of patient data, securely accessed, and stored, both in underpinning and leading to improvements in research and care.The authors would like to thank Specialist Nurses Amanda Watt and Joanne Stout at the National Renal Complement Therapeutics Centre, Newcastle upon Tyne for their contributions to data collection and maintaining the aHUS database.