Recent advances in single-cell technologies, particularly single-cell RNA-sequencing (scRNA-seq), have permitted high throughput transcriptional profiling of a wide variety of biological systems. As scRNA-seq supports inference of cell-cell communication, this technology has and continues to anchor groundbreaking studies into the efficacy and mechanism of novel immunotherapies for cancer treatment. In this review, we will highlight methods developed to infer inter- and intracellular signaling from scRNA-seq and discuss how they have contributed to studies of immunotherapeutic intervention in the tumor microenvironment (TME). However, a central challenge remains in validating the hypothesized cell-cell interactions. Therefore, this review will also cover strategies for integration of these scRNA-seq-derived interaction networks with existing experimental and computational approaches. Integration of these networks with imaging, protein secretion measurements, and network analysis and mathematical modeling tools addresses challenges that remain with scRNA-seq to enhance studies of immunosuppressive and immunotherapy-altered signaling in the TME. Single-cell technologies provide the resolution necessary to study intercellular heterogeneity. Specifically, single-cell RNA-sequencing (scRNA-seq) permits whole-transcriptome profiling of individual cells , which sWhile scRNA-seq has been applied to a broad range of biological contexts, it has become an important tool to probe the tumor microenvironment (TME). The TME is a heterogeneous and constantly evolving milieu made up of populations of different cell types, cell-surface and secreted signals, and extracellular matrix , 6. Thesvia “immune checkpoints”  being used by over 500 users per month as of July 2020Despite the progress posed by the aforementioned methods, scalable application to existing scRNA-seq datasets remained a challenge as user-friendly platforms for construction of interaction networks did not exist. CellPhoneDB , 29 presin vitro co-culture models  as enhanced in non-responding and resistant patients. As TIM3 has been known to inhibit CD8+ cytotoxic activity and therefore can contribute to tumor progression  is the current standard of care for immunotherapeutic intervention for many cancers . Over tigression , these sIt is worth noting additional open-source packages for inference of cell-cell communication from scRNA-seq that use algorithms similar to CellPhoneDB. Network Analysis Toolkit for Multicellular Interactions (NATMI)  quantifiCellPhoneDB in particular has advanced the study of intercellular signaling in the tumor microenvironment from scRNA-seq with its scalable and accessible platform. However, functional cell-cell communication extends beyond the interaction of extracellular ligand and receptor, as signaling cofactors can upregulate or antagonize downstream signal propagation, and ligands from “sender” cells can influence intracellular signaling cascades and target gene expression in “receiver” cells.CellChat  is an alFirst applied to the wound healing microenvironment, CellChat has shown promise in the characterization of malignant and immunotherapy-altered signaling in glioblastoma , esophagOther recently published algorithms NicheNet  and CytoThe use of a prior model with ligand-to-intracellular target gene axes to infer cell-cell communication from scRNA-seq data represented a departure from the interaction scoring approach matching ligand and receptor expression adopted by most preceding methods. Algorithmically, preceding methods follow a common procedure  and NATM+ mature cDC subset enriched for immunoregulatory genes (mregDCs). Cheng et al. were keen to understand mregDC development, as this novel DC subset has been shown to originate from both cDC1s and cDC2s, resulting in different roles in anti-tumor immunity. Using NicheNet, IL-4 and IL-12 were identified as drivers of the cDC1-derived mregDC signature, while IL-15 and IFNs were linked to the cDC2-derived mregDC signature   , 111 andMERFISH) , have aiMERFISH) . IntegraMERFISH) , the intMERFISH) , linkingin situ barcoding method for multiplexed profiling of a predefined panels of RNAs or proteins from fixed tumor tissues  was found to correlate with prolonged progression-free survival and overall survival in patients. Similar to MERFISH, GeoMX DSP thus provides high resolution imaging of focused regions of the TME, supporting local rather than global evaluations of ligand, receptor, and cell type co-localization.GeoMX DSP is an  tissues . This me tissues , non-sma tissues  and head tissues . In melain situ hybridization methods like MERFISH and sequential fluorescence in situ hybridization (seq-FISH+)  is a microfluidics-based method for 10μm-resolution multi-omic profiling of fixed tissues . By deliq-FISH+)  by takinq-FISH+) ], DBiT-sq-FISH+) . Taken tin vivo dynamics and migration in tumor models at single-cell resolution. Following surgical implantation of an imaging window, live fluorescent imaging of the TME can be collected, generally over a period of hours to days. Luthria et al.  played in anti-inflammatory TAM polarization. Using the cell-cell communication inference strategy from Kumar et al. (in vitro (in a co-culture model system) and in vivo. The cell-cell interaction networks inferred from scRNA-seq therefore directly provided an experimentally testable target for immunotherapeutic intervention in this study, although the authors did primarily focus on crosstalk between immune cell populations in these hypothetical communication networks. Investigation of TAM-tumor cell crosstalk as inferred from scRNA-seq was not presented in this work. The focus of their IVM data was to demonstrate physical interactions between TAMs and tumor cells in the TME and pinpoint these interactions as a catalyst for downstream alterations in microtubule dynamics. Reanalysis of the scRNA-seq data to instead concentrate on inferring extracellular signaling events between TAMs and tumor cells has the potential to identify additional mechanisms of cancer cell migration and anti-inflammatory TAM polarization for therapeutic targeting with direct support from the IVM work.Beyond SRT methods, an array of complementary imaging techniques for both dynamic and static receptor-ligand and sender-receiver cell co-localization has been developed and used in the TME. Intravital microscopy (IVM)  has emera et al.  showcaser et al.  and scRNr et al. . Treatme+ cytotoxic T cells and dendritic cells (DCs), producing IFNγ and IL-12 signals, respectively, upon treatment with anti-PD-1. This T cell-DC crosstalk was thus found to form a positive feedback loop resulting in stimulation of anti-tumor T cell immunity that was indirectly downstream of anti-PD-1 binding. Garris et al.  is under development , integrates subnanoliter microchambers with high-density antibody barcode microarrays for simultaneous detection of up to 15 cytokines from over a thousand single cells in parallel. Beyond fundamental studies of macrophage proinflammatory dynamics in vitro  isolated from 8-week-old murine melanoma tumors , and it has been primarily tuned to investigate T cell secretion. Commercialization has facilitated access to this technology by over 100 pharmaceutical companies and medical centers worldwide+ and CD8+ T cell subsets, as measured by the SCBC, was identified as a distinguishing factor between response and resistance to anti-PD-1. The commercialized device has also been used to understand the functional response to other T cell-targeted treatments, including chimeric antigen receptor (CAR) T cell therapy (While the SCBC has proven its utility in measuring secretion from tumor-associated myeloid cells (namely macrophages and DCs), an up-to-42-plex version of this device has been commercialized with IsoPlexis , weighted , and densely connected, data mining and biological interpretation are complicated. However, they also present an opportunity to use concepts from graph theory and mathematical modeling to identify the most central, influential, or biologically relevant signaling axes for experimental validation.Graph theory is a field of mathematics that views networks as a collection of nodes, or vertices, connected by edges. In the context of scRNA-seq-derived interaction networks, cell types or subtypes would be considered nodes, with ligand-receptor or ligand-target gene axes comprising the edges between them. Centrality is a graph theory concept concerned with identifying “important” nodes in a graph, where the definition of importance changes depending on the “angle” at which the graph is analyzed. Measures of centrality include degree, betweenness, closeness, pagerank, and eigenvalues. Degree is the simplest measure of centrality, counting the absolute number of edges pointing toward  and away from  a node. Betweenness quantifies a node’s influence on the flow of information in the network, which can identify “bottlenecks” or nodes that serve as “ambassadors” between modules of signaling. Similarly, closeness indicates how directly or indirectly each node in a graph can communicate with one another. Pagerank is a measure of “popularity,” as it answers the question of how often a particular node is communicated through during a random walk on the graph. Finally, eigenvalue centrality is similar to degree centrality in that it quantifies a node’s influence through its connections with others. However, this metric is better suited to a weighted network, as it takes strength of communication and influence of other nodes into account.While centrality metrics are often reserved for analyzing scRNA-seq-derived gene regulatory networks , 147, thOutside of graph theory, regression modeling presents an opportunity to interpret scRNA-seq-derived cell-cell communication networks with respect to relevant quantitative and qualitative biological behaviors, including tumor outcome, tumor growth rate, and immune cell infiltration. Elastic net regularization  is a regscRNA-seq has proven to be a powerful tool for high throughput prediction of cell-cell communication. Transcriptome-wide profiling of genes encoding for ligands, receptors, intermediary signaling proteins, and signaling targets, in conjunction with novel algorithms like CellPhoneDB, CellChat, NicheNet, and CytoTalk, has characterized signaling mechanisms underlying homeostasis and disease across biological systems. In particular, scRNA-seq-derived interaction networks have facilitated identification of immunosuppressive and immunotherapy-altered cell-cell signaling in the TME, which contribute to the design and development of more effective immunotherapeutic interventions in the clinic. Validation of these networks will only serve to strengthen the hypotheses that they generate. Integration with SRT and other imaging technologies will help to define which interactions are physically possible in the microenvironment through co-localization of both communicating cell types and paired ligand, receptor, and signaling intermediaries. Functional measurement of protein secretion will confirm outgoing communication from “sender” cells and organize broader cell populations into relevant subsets based on patterns in secreted signaling. Further computational analysis of interaction networks by borrowing from the fields of mathematics and statistics will focus the search for experimentally targetable signaling mechanisms underlying aberrant behaviors. Together, integration of scRNA-seq-derived communication networks with powerful experimental and computational tools will expand our understanding of the role that cell-cell communication plays in emergent biological behaviors in the TME and beyond, improving outcomes for patients across malignancies.KB and KM-J contributed equally to all aspects of this article. All authors read and approved the final manuscript.This work was supported by the U.S. National Institutes of Health (U01CA238728 to KM-J).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Dynamical systems models for controlling multi-agent swarms have demonstrated advances toward resilient, decentralized navigation algorithms. We previously introduced the NeuroSwarms controller, in which agent-based interactions were modeled by analogy to neuronal network interactions, including attractor dynamics and phase synchrony, that have been theorized to operate within hippocampal place-cell circuits in navigating rodents. This complexity precludes linear analyses of stability, controllability, and performance typically used to study conventional swarm models. Further, tuning dynamical controllers by manual or grid-based search is often inadequate due to the complexity of objectives, dimensionality of model parameters, and computational costs of simulation-based sampling. Here, we present a framework for tuning dynamical controller models of autonomous multi-agent systems with Bayesian optimization. Our approach utilizes a task-dependent objective function to train Gaussian process surrogate models to achieve adaptive and efficient exploration of a dynamical controller model’s parameter space. We demonstrate this approach by studying an objective function selecting for NeuroSwarms behaviors that cooperatively localize and capture spatially distributed rewards under time pressure. We generalized task performance across environments by combining scores for simulations in multiple mazes with distinct geometries. To validate search performance, we compared high-dimensional clustering for high- vs. low-likelihood parameter points by visualizing sample trajectories in 2-dimensional embeddings. Our findings show that adaptive, sample-efficient evaluation of the self-organizing behavioral capacities of complex systems, including dynamical swarm controllers, can accelerate the translation of neuroscientific theory to applied domains. In this framework, a task-dependent objective function signifies the output performance of the complex underlying model, and the optimizer traces parameter-space trajectories of candidate points from acquisition functions operating on a simpler surrogate model. The typical surrogate model is a Gaussian process that populates the parameter space of interest with multivariate normal distributions and which serves as a prior distribution for candidate-point updates . BayesiaThe collective behavioral states of some swarming models are tractable to linear analysis of stability, density, and clustering properties –32. HoweNeuroSwarms framework for modeling emergent high-level navigation and foraging in a brain-inspired multi-agent metacontroller , with better task performance closer to zero.We constructed an objective function to evaluate the performance of the example NeuroSwarms model  in a timLH and LT as calculated in xi is indicated by the objective value Y, computed as the averageTo account for the generalizability of spatial task performance across distinct environmental geometries, each simulation-based sample constitutes play-throughs of both the Hairpin and Tunnel mazes, respectively providing loss values 2.5.Y  [The surrogate model hyperparameters were tuned by first computing the marginal log-likelihood (MLL) of the Gaussian process applied to observed parameters -BFGS-B) . The fit2.5.1.M was computed followingM selected for similar neighborhoods of parameter points as in previous training epochs. These convergence metrics determined hyperparameter convergence and enabled the resulting Gaussian process surrogate model to efficiently adapt to the NeuroSwarms parameter space.This hyperparameter tuning process described above was repeated until convergence according to two metrics: maximum posterior variance and minimum candidate dissimilarity. First, maximum posterior variance for training epoch 2.6.p-dimensional parameter points. A 2D UMAP projection allows these point clusters to be simply visualized as images or scatter plots, for which the x-axis and y-axis constitute an arbitrary coordinate frame. For UMAP scatter plots, as in yi   result fas in yi , and ass3.3.1.We demonstrate Bayesian optimization methods see  for tuni3.2.p = 9 dynamical NeuroSwarms parameters , q-batched Noisy Expected Improvement (qNoisyEI), or random parameter sampling , as shown in t = 41.02 s). Reward-capture speed using the default parameters was additionally exacerbated in the Tunnel maze (t = 175.42 s). In contrast, the qEI-tuned swarm captured all three rewards (t = 34.88 s). We attribute the worse performance of the hand-tuned default parameters to longer dynamical time-constants and thus slower behavioral responsivity. Thus, compared to manual parameter tuning for each maze environment, our Bayesian batch-optimization process  cooperative foraging without directly modifying NeuroSwarms’ underlying mechanisms. In general, this feature allows a task-dependent objective to evaluate multi-agent performance in collective tasks involving, e.g., social coordination or distributed consensus. In contrast to the regular but fragmented geometry of the Hairpin maze (t = 5.46 s) vs. R3 (t = 31.78 s) reflects the characteristic time-scale differences between coordinated reward-approach trajectories and exploratory swarming trajectories, respectively. Comparing the pre-capture  and post-capture  trajectories for each reward  and t = 31.78 s). Additionally, the default parameters from Monaco et al. (2020) [κ = 6.6), but weak swarming-based exploration . This combination of behavioral forces increased the time-to-capture for all five rewards. Thus, we attribute slow reward-capture to a combination of longer dynamical time-constant parameters and exploration–exploitation mismatches. Moreover, if the energy budget of agent locomotion  were to be taken into account by the objective function, a slower behavioral repertoire enabled by these parameter regimes could help to minimize energetic or inefficient navigational patterns.Trained acquisition functions can be used to predict the performance of unobserved regions of the parameter space. To test predictive selection, we generated 500 samples from the qEI acquisition function and the posterior distribution of its trained Gaussian process surrogate model. The qEI sample means from the posterior . In part. (2020)  entailed4.Neuroscience-inspired learning and control methods have seen increased interest from robotics, artificial intelligence, and multi-agent control. Here, we presented a demonstration of exploring and visualizing the parameter space of a multi-agent model with complex dynamical behaviors using sample-efficient Bayesian optimization with Gaussian process surrogate models. We introduced an objective function for a spatial cooperative foraging task in NeuroSwarms simulations  to prediBy learning UMAP embeddings , we demop up to ~20—Bayesian optimization may facilitate adaptive and efficient computational exploration of dynamical parameter spaces, resulting in the identification of distinct and complex system behaviors.As parameter size grows, the computational cost of the matrix inversions required to calculate updated Gaussian process parameters increases exponentially and eventually outweighs the gains in adaptive search efficiency provided by computing the acquisition function over the surrogate model to advance the sample trajectory . This liFuture work is needed to develop new controller models and critical spatial tasks to explore the capabilities of multi-agent objective functions that adapt efficiently to the characteristics of diverse environments . We theorize that heterogeneous variation of swarm spatial structure and intertemporal coordination dynamics will be able to support a form of swarm metacognition that allows adjustment to the available goals in an environment, without initial knowledge of the goals or their locations. This approach could extend the flexibility of Bayesian optimization to operate in diverse environments and adapt efficiently to tasks with difficult or uncertain goals.


For over two decades, the EBMT has updated recommendations on indications for haematopoietic cell transplantation (HCT) practice based on clinical and scientific developments in the field. This is the eighth special EBMT report on the indications for HCT for haematological diseases, solid tumours and immune disorders. Our aim is to provide general guidance on HCT indications according to prevailing clinical practice in EBMT countries and centres. In order to inform patient decisions, these recommendations must be considered in conjunction with the risk of the disease, risk of HCT procedure and non-transplant strategies, including evolving cellular therapies. HCT techniques are constantly evolving and we make no specific recommendations, but encourage harmonisation of practice, where possible, to ensure experience across indications can be meaningfully aggregated via registry outputs. We also recommend working according to JACIE accreditation standards to maintain quality in clinical and laboratory components of practice, including benchmarking of survival outcomes. Since the last edition, the COVID-19 pandemic has affected clinical decision making and activity across indications. Although the full impact of the pandemic is yet to be determined, we recommend that decision making across indications is delivered with ongoing reference to EBMT and national COVID-19 guidance, in accordance with current local conditions. This is the eighth report from the European Society for Blood and Marrow Transplantation (EBMT) covering indications for haematopoietic cell transplantation (HCT) according to prevailing clinical practice in EBMT countries and centres . For ovehttps://www.ebmt.org/covid-19-and-bmt). Consequently, there has been a temporary reduction in HCT rates in some indications, with necessary prioritisation between indications and delays in treatment that may have impacted upon outcomes of HCT. Although the full impact of the pandemic is yet to be determined and our understanding and evidence-base is evolving, we recommend that decision making across indications is made by an MDT with reference to EBMT and other national guidance in relation to COVID-19, and in accordance with current local conditions.Since the last update, we have experienced the Coronavirus disease-19 (COVID-19) pandemic, which has affected HCT activity for a variety of reasons, from risks to patients, availability of donors and stem cell products, through to maintenance of staffing and services . Waves oThis guidance does not primarily intend to cover cellular therapies, but makes reference to the application of CAR-T cells as a treatment option alongside HCT in acute lymphoblastic leukaemia (ALL) and lymphomas, and, more developmentally, in multiple myeloma, chronic lymphocytic leukaemia and acute myeloid leukaemia.Importantly, we recognise that there is an overlap between adult and paediatric indications, particularly in the ‘teenager and young adult’ (TYA) group, and definitions of paediatric and TYA (and therefore ‘adult’ care) vary internationally. Despite the age cut-offs, which are also influenced by EBMT registry definitions, the indications should be interpreted with flexibility, particularly in the TYA age group and some ‘paediatric’ and TYA indications may occasionally extend into older adult age groups. Given this consideration, we have combined ‘Inherited diseases’ into considerations for all ages, although the predominant use of HCT will be for the paediatric age group.HCT refers to any procedure where haematopoietic stem cells (HSC) of any donor type and source are given to a recipient with the intention of repopulating and replacing the haematopoietic system in total or in part. HSC for HCT can be derived from bone marrow (BM), peripheral blood (PB) or cord blood (CB).Donor type is categorised as autologous, syngeneic and allogeneic, the latter being either related or unrelated. Beyond HLA-matched related  donors (MSD), a well-matched unrelated donor (MUD) is defined as a 10/10 identical unrelated donor (UD) based on HLA high-resolution typing for class I  and II . A mismatched unrelated donor (MMUD) refers to a UD mismatched in at least one antigen or allele at HLA-A, -B, -C, -DR or -DQ and a haploidentical donor refers to one haplotype mismatched donor, most frequently being a family donor. Criteria for UD selection have been proposed , 4, but Recent developments include non–T-cell-depleted haploidentical HCT, mainly with post-transplant cyclophosphamide (PTCy) as graft-versus-host disease (GVHD) prophylaxis that has led to a reduction in chronic GVHD and TRM  in this Transplant decisions derive not only from sound clinical and scientific evidence but also from each centre’s research priorities, local expertise, cost considerations and ease of access to particular transplant modalities, along with individual patient and donor preference. Ultimately, we recommend all aspects are brought together and documented in an MDT meeting.EBMT indications are classified into four categories Table , to descThe updated 2022 classification of HCT procedures in adults is shown in Table As per EBMT’s last activity survey, AML accounts for more than one-third of allo-HCT transplants . The hett, inv(16) or t and NPM1 mutation plus wild-type FLT3-ITD or FLT3 with low-allele ratio (<0.5)  [Overall, allo-HCT in first complete remission CR1) is recommended for adverse and intermediate-risk AML as defined by the European LeukemiaNet (ELN) risk stratification that is based on cytogenetics and mutational phenotype, while HCT is not recommended for AML patients with favourable disease  is recom. Specifiversial) .Allo-HCT may be effective in a subset of patients with primary refractory AML (defined as failure to achieve CR after two cycles of induction chemotherapy) and relapsing AML , especiaAML patients in the favourable risk category may be considered for auto-HCT especially if they are MRD negative post induction therapy , 14, whiAs per EBMT’s latest activity survey, ALL is the second most common indication for allo-HCT, accounting for 17% of cases , with PhResults of allo-HCT from UD are constantly improving as those from non–T-cell-depleted haploidentical donors, as alternatives for transplant-eligible ALL patients lacking a MSD , 40. Impt, complex karyotype  and Ph-positive ALL.High-risk cytogenetics at diagnosis is an indication for allo-HCT, even in MRD-negative patients, at least until additional studies are available –52. AdveAllo-HCT is also indicated for adult ALL patients in CR2 or beyond  and may Anti-CD19 CAR-T cell therapy is approved for patients up to 25 years of age based on the ELIANA study , and morRelapse after CAR-T cell therapy occurs in 30–50% of patients (with CD19 negative relapse in up to 40%) , 61. TheFor Ph-positive ALL, allo-HCT is still indicated in CR1 . Post-trIn contrast to B-ALL, salvage options in T-ALL are limited and consolidation with HCT in CR1 should be considered in high-risk patients. Similarly to B-ALL, MRD is a key predictor of relapse , 67. OthWith respect to auto-HCT, in the donor versus no donor studies and subsequent meta-analyses, no beneficial effect was observed –37. The Since the advent of TKIs, allo-HCT is not recommended as a first-line treatment in CML. In the vast majority of CML patients in chronic phase (CP), imatinib or second-generation TKIs, such as dasatinib, nilotinib or bosutinib, should be the first-line therapy. Some patients achieving molecular remission with TKI treatment have remained in molecular remission for long periods after cessation of the drug and complete discontinuation of TKI could be obtained in about 40%, but it remains to be seen in the long term whether these patients are cured , 72.Patients who fail first-line therapy according to ELN guidelines should start on second-line TKI therapy. Donor search should start in patients who fail two lines of TKI. They should receive treatment with third-line TKI depending on ABL mutation analyses and are candidates to proceed to allo-HCT as soon as possible whilst in optimal response if EBMT risk score is 0–1 or, in the case of a prior loss of cytogenetic or haematological response to second-line TKI, if EBMT risk score is 0–4. If there is no haematological response to second-line treatment, patients are candidates for allo-HCT with any EBMT risk score. Patients with ABL mutations resistant to third-generation TKI or with T315I mutation are candidates to undergo HCT with any EBMT risk score after failing second or third-line TKI.Patients in advanced phase CML referred for an HCT could receive TKI or TKI± intensive therapy as preparation for HCT, which should be performed as soon as possible after achieving CP2 without the need for deep cytogenetic or molecular responses.Patients with a syngeneic donor are always candidates for HCT with standard conditioning. Auto-HCT is generally not recommended outside clinical trials.Overall, the main goal of CML therapy should be to prevent progression to the advanced phase or >CP1, as outcomes post-transplantation are worse in those situations. Patients with CML should be followed closely and considered for HCT while still in CP1 if they fulfil the criteria described above.Allo-HCT remains the only potential curative option for patients with MPNs. However, polycythaemia vera and essential thrombocythaemia are generally not indications for allo-HCT unless the disease has progressed to secondary myelofibrosis or secondary leukaemia –75. Due The introduction of JAK inhibitors in the treatment of primary myelofibrosis may help to improve constitutional symptoms and to reduce spleen size before HCT, but their definitive role needs to be determined . Recent Auto-HCT for MPN is generally not recommended outside clinical trials.Allo-HCT is a curative option for patients with higher-risk MDS, but patient-related factors, especially age and co-morbidities, often impact upon feasibility of HCT. EBMT proposed a transplant-specific risk score for MDS patients , which, 9/L, platelet count <50 × 109/L), or severe BM fibrosis should be considered for allo-HCT. Otherwise, patients in this risk group should be offered non-transplant options as first line of therapy [While allo-HCT is a standard of care in suitably fit patients with higher-risk MDS, decision making becomes more challenging for the intermediate IPSS-R group, where patients have a median survival of 3 years without treatment and 25% AML progression at 3.2 years. Patients in the intermediate IPSS-R risk group with >5% marrow blasts at diagnosis, poor karyotype, profound cytopenias  [According to the 2016 WHO classification of myeloid neoplasms, CMML is classified in three stages: CMML-0  and CMML-2 (blasts 10–19% in BM or 5–19% in PB). There are two variants of the disease, ‘dysplastic’ and ‘proliferative’, depending on the circulating leukocyte count (≤13 × 10ctively) .For the evaluation of disease-related factors, the use of a CMML-specific prognostic scoring system (CPSS) is recommended, possibly chosen among those including molecular information  .Similar to MDS, and despite the absence of evidence from prospective and retrospective trials, pre-transplant treatment with hypomethylating agents or intensive chemotherapy is generally recommended for CMML-2 and CMML with severe proliferative features and/or extramedullary infiltration not controlled by conventional cytoreductive drugs, whereas upfront transplantation may be the preferred strategy for patients with low blast percentages. Recently, a ‘CMML transplant score’, including ASXL1 and/or NRAS-mutated genotype together with BM blasts >2% and co-morbidities, has been proposed to facilitate personalised counselling for CMML candidates for allo-HCT .The introduction of signalling pathway inhibitors (PIs), such as the Bruton’s TKI, ibrutinib, the phosphatidylinositol-3-kinase inhibitor, idelalisib, or the BCL2-inhibitor venetoclax, has changed management algorithms and HCT indications in CLL. EBMT and ERIC (European Research Initiative in CLL) have proposed a revised definition of high-risk CLL driven by TP53 abnormalities and response to treatment with PIs . PatientPatients with CLL and a concomitant MDS and those with clonally related aggressive transformation of CLL should be considered for allo-HCT regardless of treatment stage of their CLL .Auto-HCT is generally not recommended in CLL. However, it could be considered a clinical option in patients with a histological transformation clonally unrelated to CLL .Since December 2015, lymphoma patients are reported to EBMT via the mandatory MED-A as being in ‘true’ CR1 (first CR directly by standard first-line treatment), or as being in ‘first’ CR , clearly segregating patients with different prognoses. Therefore, the recommendations for lymphoma  regarding treatment options within the scope of this guideline, and includes peripheral mediastinal B-cell lymphoma, high-grade B-cell lymphoma NOS (not otherwise specified) and double-hit and triple-hit lymphomas.The anti-CD19 CAR-Ts, axicabtagene ciloleucel and lisocaptagene maraleucel, show significant improvement in PFS and a strong trend in OS  in two Phase III clinical studies in high-risk r/r LBCL compared with salvage therapy (ST) followed by auto-HCT , 94. HigIn late chemosensitive relapse of LBCL after first-line therapy including rituximab, auto-HCT remains standard of care, although CAR-Ts also have to be considered for these patients. As real-world data with anti-CD19 CAR-Ts in third-line use  have conFor patients failing second-line salvage therapies, relapsing after auto-HCT or with refractory disease, allo-HCT remains a clinical option –103 afteThe recommendations for primary central nervous system (CNS) lymphoma are maintained. In this DLBCL subset, there is evidence from a number of non-comparative trials and two randomised controlled trials (RCT) that consolidating auto-HCT in first remission is safe and effective, justifying categorisation as S/II –109. TheIn the era of therapeutic antibody maintenance, evidence for benefit of HCT in CR1 is lacking in patients with untransformed FL and in those with high-grade transformation who have not received systemic treatment for the underlying FL before the histological transformation. In contrast, consolidation with auto-HCT may be a clinical option in patients with the chemosensitive high-grade transformation of FL, if they had received prior systemic treatment, especially immunochemotherapy. Beyond the potential efficacy of CAR-T in FL, novel drugs, such as idelalisib, have not changed the natural history of the disease, and transplant indications for FL beyond CR1 remain unchanged compared to previous editions , 112.The anti-CD19 CAR-T axicabtagene ciloleucel results in a high number of durable responses in the third and higher treatment line in a non-comparative clinical trial . It is tWith the advent of more effective novel agents for WM, such as rituximab, purine analogues, proteasome inhibitors, and kinase inhibitors, strategies using first-line auto-HCT are increasingly questionable and are not recommended outside clinical trials . Auto-HCIbrutinib has been approved as an effective salvage treatment for patients with relapsed or refractory MCL. However, in the RCT setting progression-free survival of relapsed MCL is modest with ibrutinib . MoreoveIn a non-comparative study, the anti-CD19 CAR-T, brexucabtagene autoleucel, showed a high number of durable responses in patients failing at least two prior therapies including ibrutinib and mostly pretreated with auto-HCT . The difPeripheral T-cell lymphomas usually carry a very poor prognosis. Allo-HCT is effective in patients with relapsed and refractory disease and recommended as standard of care in patients with chemosensitive relapse, as the only curative modality in this condition. In CR1, however, a prospective randomised trial testing superiority of allo-HCT over auto-HCT was prematurely terminated due to the low likelihood of meeting its primary endpoint . Thus, bPrimary cutaneous T-cell lymphomas in early stage have an excellent outcome, and HCT is generally not recommended. However, patients with EORTC/ISCL advanced stages IIB to IV have a dismal prognosis with conventional therapy –126. AllTargeted agents such as brentuximab vedotin and checkpoint inhibitors may shift the transplant algorithms for HL in the future. For now, as in previous recommendations, auto-HCT remains standard of care for patients with relapsed HL chemosensitive to ST, and allo-HCT in those after a failed prior autograft –134. CARThe development of new agents, such as proteasome inhibitors, immunomodulatory drugs and mAbs, have been major advances and may eventually change the position of HCT in the treatment strategy. Currently, first-line auto-HCT is still the standard of care for newly diagnosed MM patients –139. AltAs the vast majority of patients still relapse after auto-HCT, the use of a further, or ‘salvage’ auto-HCT after re-induction therapy is an option and may be of particular benefit in patients achieving a long treatment-free interval of at least 18–24 months after the first transplant . RelapseAllo-HCT is a treatment with curative potential but associated with considerable TRM and might be used in selected high-risk patients . The comTM) is the first cell-based gene therapy approved by the FDA for adults with relapsed/refractory MM after four or more lines of therapy, including an immunomodulatory agent, a proteasome inhibitor, and an anti-CD38 mAb. In addition, EMA has recommended granting conditional marketing authorisation in the European Union for ABECMATM for the treatment of adults with relapsed/refractory MM who have received at least three previous therapies, including an immunomodulatory agent, a proteasome inhibitor and an anti-CD38 mAb. Whether CAR T-cell therapy could replace auto-HCT is under investigation in randomised phase III studies .CAR T-cell therapy has shown promising results in patients with refractory/relapsed MM –157. Idet, can also guide therapy [Patients with systemic immunoglobulin-light-chain (AL) amyloidosis without severe heart failure may benefit from auto-HCT . However therapy . Allo-HC therapy .HLA-identical sibling allo-HCT is considered the standard of care for adult patients with SAA, although the outcomes decline over the age of 40 –166. In MUD HCT can be considered as a first-line choice in young patients aged <18 years based on the excellent outcome compared to historical matched controls , provideMUD HCT in young and adult patients is indicated after failure to respond to one course of IST, normally assessed at 3–6 months. Age of recipient is also an issue for MUD HCT, and along with assessment of co-morbidities and other patient and transplant characteristics , should help in evaluating patients who would benefit most from the procedure. Classically, patients aged up to 30 years within the first year from diagnosis and refractory to immunosuppressive treatment are the best candidates for MUD HCT. Otherwise, a non-transplant approach is recommended  , 168–174Alternative donors for allo-HCT  may be considered after failure to respond to immunosuppressive therapy. Excellent results have been reported especially in young patients up to 20 years of age in the absence of MSD or MUD –181. ThoConstitutional SAA and BM failure syndromes, including Fanconi anaemia (FA), dyskeratosis congenita (DKC) and other telomere diseases, may not only present in childhood but also in adults, often with more subtle clinical features. All cases of SAA being considered for HCT should have appropriate diagnostic workup, including molecular genetics, to establish or exclude constitutional BMF syndromes, with consideration of any impact on family donors and conditioning regimen (see relevant sections below).The introduction of anti-complement therapy with eculizumab changed the natural history of the disease, and allo-HCT became generally not recommended for patients with haemolytic PNH for whom eculizumab is available. Potential indications remain dependent on the individual clinical manifestations: (i) AA/PNH syndrome, that is, PNH occurring in the presence of severe BM failure with a hypocellular BM  and (ii) clonal evolution of PNH to MDS/AM , 185. PaCurrently, the EBMT Registry includes >63,000 HCT procedures in >46,000 patients with solid tumours, with >7000 procedures performed in the last 5 years. However, with the possible exception of patients with germ cell tumours (GCT), and highly-selected patients with breast cancer (BC), sarcoma and medulloblastoma, HCT is generally not recommended or developmental for most indications in solid tumours . With veDespite the encouraging role of immune surveillance and immune responses against several solid tumours –189 recoThe role of auto-HCT in BC at high-risk of recurrence and metastatic has been assessed by several randomised trials and meta-analyses of individual patient data , 193. AsIn GCT, auto-HCT is a standard of care for patients with disease refractory to platinum-based chemotherapy or with a second or further relapse, a clinical option as a second line in high-risk patients, and generally not recommended as first-line therapy –202. FinAuto-HCT, being per se capable of inducing marked and rapid tumour regression, may still represent a treatment modality for selected chemosensitive solid tumours and worthy of further study in combination with effective target agents, including immunotherapy. While awaiting results of further prospective trials, the EBMT registry remains an important source to survey indications, outcome and clinical risk factors in patients with solid tumours treated with auto- and allo-HCT.Autologous and allo-HCT represent a viable therapeutic approach for many severe AD, resistant to standard therapies, after careful balance of benefits and risks . Most trEvidence is continually evolving for auto-HCT, predominantly in MS –212, andDespite improved survival over time, allo-HCT has remained predominantly used in younger patients . AccordiMultidisciplinary guidelines and recommendations for both autologous and allo-HCT, across a range of AD, have been published by the EBMT and other professional societies to support clinicians, scientists, as well as patients and carers , 236–243While HCT in inherited diseases is predominantly performed in childhood, adult patients with inherited diseases, including haemoglobinopathies, constitutional BMF syndromes and inborn errors of metabolism (IEM) and immunity (IEI) are increasingly considered for HCT . The indAllo-HCT in children and adolescents represents over 20% of overall allo-HCT activity, with a particular use in congenital and non-malignant diseases, many of which are rare. Transplant complications in paediatric patients impact on with the vulnerabilities of the developing child, including development-related organ dysfunction including infertility, delayed hormonal development, growth retardation and a high risk for malignancies in congenital disorders with chromosomal breakage syndromes. Improvements in high-resolution HLA matching for UD, conditioning regimens and supportive care for infectious and non-infectious complications have progressively reduced mortality and encouraged the positioning of allo-HCT particularly in non-malignant indications at an earlier stage in the course of the disease with patients in a better performance status rather than as a ‘last chance for cure’. GVHD remains the major limitation for patients without optimal matched donors. New allo-HCT strategies should improve outcomes for MMAD. The updated 2022 classification of HCT procedures in children and adolescents is shown in Table Childhood AML is a rare and heterogeneous disease, with increasing rates of cure and survival with intensive chemotherapy, particularly for patients with favourable prognostic markers. Thus, allo-HCT is not recommended as front-line therapy in low-risk patients, but remains a standard of care for patients in CR1 with high and very high-risk disease with a well-matched donor –248. AltAllo-HCT from MSD and MUD is a standard of care for high-risk ALL patients in CR1 and in CR2 or later –256. WhiAs discussed earlier for adult patients, since the advent of TKI, allo-HCT is not recommended as a first-line treatment of CML in children and adolescents either. However, it remains a standard option for patients with treatment failure, recurrence after receiving salvage second-generation TKI treatment and advanced phase CML –266. Of Allo-HCT from a MSD or a MUD is the treatment of choice for children with primary MDS including juvenile myelomonocytic leukaemia, as well as secondary AML –273. AutNearly all children and adolescents with Hodgkin and non-Hodgkin lymphoma are cured with multidrug chemotherapy. Few such paediatric patients are eligible for HCT Table  274–278278, but Although the published results have not proven yet an unequivocal benefit for most indications, children and adolescents with solid tumours can undergo auto-HCT following high-dose chemotherapy as a clinical option or within research protocols, preferably as part of first-line treatment strategies Table . NeuroblAllo-HCT from an MSD is the standard front-line therapy for children with acquired SAA. In patients without an MSD, a well-matched unrelated HCT is now also considered a standard front-line therapy in many patients if the donor is readily available, and the search should in any case be initiated before starting any immunosuppressive therapy –285. ForAutologous and allo-HCT may be considered a clinical option for children and adolescents with AD , 236. OvAuto-HCT may be considered for carefully selected subpopulations of patients with juvenile inflammatory arthritis  and other ADs including systemic sclerosis, systemic lupus erythematosus, vasculitis and polymyositis-dermatomyositis. Paediatric multiple sclerosis is a rare indication of auto-HCT, and long-term responses have been reported .Crohn’s disease is a potential indication for auto-HCT. However, there should be careful consideration of monogenic forms of inflammatory bowel disease , lysosomal storage diseases (LSD), and some other IEM disorders . Among LIn metachromatic leukodystrophy (MLD), a central and peripheral demyelinating disease, allo-HCT is ineffective in early infantile disease but may have an impact in attenuating juvenile and adult forms when applied early in the disease course. Allo-HCT also has a role in other LSD, such as MPSII, MPSVII and Wolman disease. Given the rarity, expert MDT assessment of individual cases is essential.Among PSD, allo-HCT is effective at preventing disease progression in the childhood, or rarely adult, presentation of cerebral inflammatory disease of X-linked adrenoleukodystrophy (X-ALD). Not all males within kindreds are affected, and allo-HCT should not be offered to those with advanced disease, as it further likely has no impact on other disease manifestations including the later development of myelopathy. Typically, genetically affected males are screened with serial MR imaging and transplant is offered at the earliest sign of disease.Mitochondrial neurogastrointestinal encephalomyopathy is a rare multisystem disorder caused by mutations in thymidine phosphorylase . Allo-HCGene therapy from gene-corrected autologous HSC is very promising in IEM through providing supra-physiologic enzyme production. This is now established therapy in late infantile MLD and trials are in progress in MPSIH and MPSIIIA . LentiviAllo-HCT from a healthy MSD or a related CB represents the treatment of choice for young patients with transfusion-dependent ß-thalassaemia (TDT). For patients without an MSD, a transplant from a MUD is a clinical option –324.Li et al.  showed iThe situation in sickle cell disease (SCD) is slightly different to TDT. The mortality of infants and small children with SCD has reduced significantly with simple measures of conventional therapy such as vaccination, antibiotic treatment, parent education and the use of hydroxyurea starting in infancy. Nevertheless, adult mortality has only shifted to older ages as conventional therapies do not have an impact on progressive organ damage. Therefore, adults continue to succumb to heart, pulmonary, renal failure and stroke, and remain often disabled many years prior to these terminal events. For this reason, allo-HCT from a MSD is standard of care and should be offered prior to the emergence of serious complications –331.In SCD, age is a significant denominator for outcome since the incidence of acute and, more significantly, chronic GVHD reaches 17 and 20%, respectively, following HCT from a MSD in patients beyond 15 years . The genIn SCD, MSD/MUD donor availability is below 20%. Recent reports demonstrated successful outcomes with haploidentical transplantation , 335 andSince SCD is mainly a systemic vasculopathy with unexpected and disease-specific complications during allo-HCT such as PRES, macrophage activation syndrome, significant pain crises during HCT and organ failure, experimental approaches should only be performed in controlled clinical trials in highly experienced centres .CRISPR-Cas9 gene-editing approaches are currently evaluated in Phase I trials as potential alternatives to allo-HCT . LentiviImportantly, HCT for hemoglobinopathies should be performed early in life to reduce complications, in particular the incidence of chronic GVHD. Delay may also lead to irreversible damage due to iron overload in patients with TDT and systemic vasculopathy in patients with SCD.OP is a heterogeneous genetic disease related to several gene defects affecting osteoclast function and characterised by impaired bone resorption. Bone density is increased on X-ray. Classical infantile OP leads to early manifestations such as macrocrania, vision impairment, haematological insufficiency, hepatosplenomagaly and hypocalcemia. Urgent HCT is indicated after exclusion of neurodegenerative and osteoclast-extrinsic defects. Atypical, delayed disease may occur, for whom HCT may be considered in case of haematological insufficiency or imminent visual impairment .Currently, the JACIE standards, produced in collaboration with the Foundation for the Accreditation of Cellular Therapy (FACT), are in their 8th edition and have been expanded to incorporate immune effector and other cell and gene therapies, such as CAR-T cells. JACIE has been increasingly recognised by governmental bodies and competent authorities in several EU member states and extends beyond Europe with accredited centres worldwide. Importantly, JACIE accreditation appears to have an impact on survival outcomes and donor safety , 338–344In terms of ongoing developments, JACIE and the EBMT Registry have delivered the first two pilot phases of a risk-adapted benchmarking system that takes into consideration the heterogeneity of the disease, patient and transplant characteristics. This will enable EBMT member centres to benchmark their survival outcomes against national and/or international norms, irrespective of the size of their HCT community, and potentially this could apply to individual indications to provide ‘real-world’ estimates and benchmarking of survival at international, national or centre level and comparison with non-transplant alternatives .For over two decades, the EBMT indications reports have incorporated developments in HCT practice based on scientific and technical developments in HCT. We encourage harmonisation of practice, where possible, to ensure meaningfully aggregated experience across indications via registry outputs. We also recommend working according to JACIE accreditation standards benchmarking of outcomes to maintain quality in HCT practice.Moving forward, all treatment decisions, whether HCT or non-HCT, need to accommodate the ongoing COVID-19 pandemic. Although the full impact of the pandemic is yet to be determined, our understanding and evidence-base is evolving in combination with public health measures, vaccination strategies and new treatments in the HCT population. We therefore recommend that decision making across indications is delivered within the MDT with reference to EBMT and other national guidance in relation to COVID-19, and in accordance with current local conditions.


Cerebrovascular reactivity (CVR), an important indicator of cerebrovascular health, is commonly studied with the Blood Oxygenation Level Dependent functional MRI (BOLD-fMRI) response to a vasoactive stimulus. Theoretical and empirical evidence suggests that baseline cerebral blood flow (CBF) modulates BOLD signal amplitude and may influence BOLD-CVR estimates. We address how acquisition and modeling choices affect the relationship between baseline cerebral blood flow (bCBF) and BOLD-CVR: whether BOLD-CVR is modeled with the inclusion of a breathing task, and whether BOLD-CVR amplitudes are optimized for hemodynamic lag effects. We assessed between-subject correlations of average GM values and within-subject spatial correlations across cortical regions. Our results suggest that a breathing task addition to a resting-state acquisition, alongside lag-optimization within BOLD-CVR modeling, can improve BOLD-CVR correlations with bCBF, both between- and within-subjects, likely because these CVR estimates are more physiologically accurate. We report positive correlations between bCBF and BOLD-CVR, both between- and within-subjects. The physiological explanation of this positive correlation is unclear; research with larger samples and tightly controlled vasoactive stimuli is needed. Insights into what drives variability in BOLD-CVR measurements and related measurements of cerebrovascular function are particularly relevant when interpreting results in populations with altered vascular and/or metabolic baselines or impaired cerebrovascular reserve. Cerebrovascular Reactivity (CVR), the cerebral blood flow (CBF) response to vasoactive stimuli, reflects the regulatory ability and health of the cerebrovasculature . Using BIn pathological conditions, the capacity for a vessel to dilate in response to a stimulus can be diminished due to a pre-dilated baseline, for example, in sickle cell disease bCBF is shown to be elevated and CVR diminished . In stenVariability in CVR in healthy young populations, modulated by genetic risk factors, could be a significant predicter of neurological pathology in later life . TherefoHere, we add to the small body of literature assessing bCBF and BOLD-CVR relationships, and address how key CVR data acquisition and modeling choices affect this relationship. Specifically, unlike most previous literature, we investigate the relationship between naturally varying bCBF and BOLD-CVR, in a healthy young population, and not in situations where bCBF has been artificially altered or in the presence of aging or pathology. We characterize both the between-subject relationship between bCBF and CVR in gray matter (GM), and the within-subject spatial relationship of bCBF and BOLD-CVR across different cortical brain areas.2 (PETCO2) and BOLD-fMRI, and improved our confidence in these voxel-wise CVR estimates. The breathing tasks evoke large, transient changes in the arterial blood CO2 pressure, which consequently changes blood flow; the PETCO2 measurement is our surrogate measure of arterial blood CO2 changes  between the PETCO2 regressor and the BOLD signal, by shifting the PETCO2 regressor in time to optimize the full model fit  and physiological factors . It is therefore important to estimate the CVR amplitude alongside a correction for variable CVR timing, on a regional basis  increas changes . If BOLDodel fit . There ial basis . Based oal basis  and newl2, Bandwidth = 650 Hz/Px, 5 min 12 s. Three T2*-weighted gradient-echo planar datasets were collected, provided by the Center for Magnetic Resonance Research , with the following parameters: TR/TE = 1200/34.4 ms, FA = 62°, Multi-Band acceleration factor = 4, 60 axial slices with an ascending interleaved order, 2 mm isotropic voxels, FOV = 208 mm2, Phase Encoding = AP, phase partial Fourier = 7/8, Bandwidth = 2290 Hz/Px. Single band reference images (SBRef) were acquired to facilitate functional realignment and masking. Two of the T2*-weighted functional acquisitions included short breathing tasks preceding an 8-min resting-state period with visual fixation, and the third acquisition only included the 8-min resting-state period  dataset was also acquired whilst the subject was at rest, with sequence parameters guided by the white paper : a backg2 and O2 pressures (in mmHg) were sampled through a nasal cannula worn by the participant, recorded at 1000 Hz with LabChart software , connected to a ML206 Gas Analyzer and PL3508 PowerLab 8/35 (ADInstruments) and synced with volume triggers from the scanner.Inspired and expired COThe data from this study unfortunately cannot be made openly available due to restrictions of the ethical approval that they were collected under. However, analysis derivatives that are not included in this manuscript may be provided, on request, within ethical guidelines.The T1-weighted file was processed with FSL’s  fsl_anatETCO2 values were identified, convolved with a canonical hemodynamic response function (hrf), and shifted ± 15 s in 0.3 s increments, then down-sampled to the TR. Multiple linear regression was performed separately for five data segments, two of which included breathing modulations and three of which only included resting-state data. These five data segments were created from the three acquisitions as illustrated in ETCO2 trace and the GM average BOLD-fMRI trace for each acquisition. The regression model consisted of mean, drift terms, 6 motion parameters, and a PETCO2 time-series. The beta-weight for the unshifted PETCO2 regressor, scaled by the fitted mean, produced CVR maps with no lag optimization . For CVR maps with lag optimization (Lag-Opt CVR), the model was run for each shifted PETCO2 regressor; parameter estimates were taken from the model with the largest full model R-squared , a Pearson’s correlation analysis was performed between GM bCBF and BOLD-CVR values. Correlation plots and statistical outputs were created with the R packages ggplot2  and ggpuSpearman’s rank) between bCBF and BOLD-CVR was computed using custom MATLAB code . The effect of data segment and lag optimization on bCBF-CVR spatial correlation values was tested with a repeated measures ANOVA, run with the R package permuco  corrected, and then compared against an alpha of 0.05 to determine significance.First, an average value of BOLD-CVR and bCBF within each atlas parcel  was computed. Second, for each analysis combination and each subject, a spatial correlation  were input to AFNI’s 3dRSFC function  kernel). This function computed maps of resting-state fluctuation amplitude normalized to the mean (mRSFA), amplitude of low-frequency fluctuation  and two significant main effects: correlation values were significantly higher for Lag-Opt versus No-Opt  = 6.09, p = 0.0385) and there was a significant effect of data segment on correlation values  = 3.84, p = 0.0117). BH + REST showed significantly higher values than RESTCDB (see p = 0.8732), and significant main effects of optimization scheme  = 8.34, p = 0.02682) and data segment  = 10.83, p = 0.00006). When replacing these outlier values with the mean value for that cell of the design, the same results were also found: no significant interaction  = 0.46, p = 0.7661), and significant main effects of optimization scheme  = 10.67, p = 0.0111) and data segment  = 7.40, p = 0.00031). Multiple ANOVAs were explored due to the challenges with removing outliers, particularly for within-subject designs.Two extreme outlier points were identified (greater than 3 × IQR below the first quartile or above the third), indicated by an asterisk in BH and RESTCDB data segments , followed by exploring reasons for the observed positive relationship between bCBF and BOLD-CVR and the resting-state metrics , and then consider the relevance and implications for fMRI research .We assessed the impact of methodological factors on the correlation between bCBF and BOLD-CVR. We present complimentary results to those in our previous manuscript , suggestFor variability between-subjects, a significant positive correlation between an average GM BOLD-CVR value and an average GM bCBF value was only found for BOLD-CVR data that included a breathing task and when analysis included optimization for hemodynamic lag effects. With influential points removed, the strength of the correlation always increased after lag-optimization, for all comparisons. For within-subject spatial correlations between bCBF and BOLD-CVR in cortical parcels, the bCBF and BOLD-CVR relationship was mostly positive (the negative correlations were only seen in resting-state data with no breathing tasks or in results without lag-optimization). At the group level, spatial correlations were significantly higher after lag-optimization of the CVR values. This suggests that these two methodological choices, the inclusion of breathing data and the implementation of lag-optimization, result in more physiologically accurate CVR estimates.ETCO2 are small in amplitude, as may occur if natural fluctuations in PETCO2 are small during resting-state paradigms, it can be hard to distinguish them from other physiological, artifactual, or neuronally-driven fluctuations that occur at the same low frequencies. Our observations in this work suggest that BOLD-CVR estimates derived from resting-state data also show a much more variable relationship with bCBF. The timing of the blood flow response to a change in the pressure of arterial CO2  will not be the same for each equipment setup, each participant, and each area of the brain due to methodological and physiological factors. As well as the data type to be modeled (resting state only vs. added breathing task), accounting for voxel-wise hemodynamic timings when estimating BOLD-CVR seems to lead to more physiologically grounded CVR values.We have shown previously  that addFor segments that contained breathing tasks, BH + REST showed more consistent patterns than CDB + REST. Specifically, for the between-subject correlations of GM average values, both BH + REST and CDB + REST showed positive correlations between GM bCBF and BOLD-CVR that increased after lag-optimization, but there was one subject outlier for the CDB + REST correlation analysis. For within-subject spatial correlations, the BH + REST segment had higher correlation values on average compared to CDB + REST (though this was not a significant pairwise difference), and a more consistent change with lag optimization: 8/9 subjects showed an increased (positive) correlation after lag optimization for BH + REST, whereas this was only the case for 7/9 subjects in the CDB + REST segment, with one of these seven subjects having nearly zero correlation after lag-optimization. It is unclear what drives these differences; it simply may be the case that we can more consistently get accurate CVR estimates with the BH + REST compared to the CDB + REST segment , and theCDB segment had significantly lower spatial correlations between bCBF and BOLD-CVR than the other two REST segments, irrespective of lag-optimization. It could possibly be due to residual vascular after-effects following the hypocapnic breathing task stimulus . We previously found no clear evidence for obvious differences in common resting-state metrics  between all three resting-state data segments  in multiple brain regions . They concluded that low and high autoregulation efficiencies exist at high and low bCBF values, respectively. This is again contrary to our results, where we see positive correlations between these metrics. There are key methodological and analytical differences between this study and ours: average values were derived from 4 brain lobes compared to the 96 cortical parcels in this study; they did not record PETCO2 values, precluding characterization of BOLD-CVR in units of%BOLD/mmHg; the BOLD response to the breath-hold was modeled with the convolved task design and hemodynamic timings were not corrected on a regional level; CBF and BOLD fMRI data were smoothed with an 8 mm FWHM Gaussian kernel whereas we did not apply any spatial smoothing except the adaptive spatial smoothing applied with FSL BASIL’s toolbox. Finally, the focus of their study was to compare sex differences in bCBF and BOLD-CVR; their CBF and CVR correlations are displayed as group averages with large standard deviations, and it is therefore hard to distinguish the contribution of individual variability to this main result.There are many studies in the literature including both bCBF and BOLD-CVR values for healthy young populations, but few studies that directly assess the covariance of these values. In conflict with our results, a PET study of healthy subjects, of similar age to this study, showed a significant  regions . This di regions  with a s regions  but no dpositive correlation between bCBF and BOLD-CVR across subjects often explain this in the context of cerebrovascular aging effects: in elderly adults, a smaller capacity for vasodilation is often seen alongside lower bCBF , in 9 healthy individuals. These metrics showed stronger and more consistent positive correlations when CVR was modeled with breathing task data as opposed to resting-state data only, and when CVR timing was accounted for. Using a vasodilatory breathing-task (BH) produced more consistent results than a vasoconstrictive breathing-task (CDB). The stronger relationship between bCBF and BOLD-CVR suggests more physiologically valid CVR estimates when such methodological elements are incorporated. Future research should investigate the basis of the observed positive relationship between bCBF and BOLD-CVR in larger normative samples and across the lifespan. Understanding how baseline vascular physiology relates to dynamic vascular processes is particularly important in clinical cohorts that present with altered vascular and/or metabolic baselines or impaired cerebrovascular reserve.molly.bright@northwestern.edu.The datasets presented in this article are not readily available because of restrictions of the ethical approval that they were collected under. However, analysis derivatives that are not included in this manuscript may be provided, on request, within ethical guidelines. Requests to access the datasets should be directed to MB, The studies involving human participants were reviewed and approved by Northwestern University’s Institutional Review Board. The patients/participants provided their written informed consent to participate in this study.RS: conceptualization, methodology, software, formal analysis, investigation, data curation, writing (OD), writing (RE), visualization, and project administration. KZ: methodology, investigation, and writing (RE). SM and CC-G: methodology and writing (RE). MB: conceptualization, methodology, software, investigation, resources, writing (RE), supervision, project administration, and funding acquisition. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Telephone-administered cognitive assessments are a cost-effective, feasible, and sometimes necessary alternative to in-person assessments. However, there is a scarcity of information in large cohort studies concerning mode effects, or differences in cognitive performance attributable to assessment method instead of underlying cognition, as a potential measurement threat. We evaluated mode effects on individual cognitive items and overall cognitive score using a population-based sample of community-living older adults aged 65-79 in the US in the 2014 Health and Retirement Study for whom interview mode was randomized (n=6825). We assessed mode differences in test means and reliability, whether mode modifies associations of cognition with criterion variables, and formal measurement invariance testing by mode. Relative to those assessed face-to-face, people assessed by telephone tended to have higher scores for memory and calculation items (0.06 to 0.013 standard deviations (SD)) and lower scores for non-memory items (-0.09 to -0.01 SD). We also found evidence that estimated cognition was significantly differentially related to IADL score depending on mode of assessment, observing a stronger association among participants completing telephone interviews. Measurement invariance testing identified the largest mode differences in memory and attention items: immediate noun recall, delayed noun recall, and serial 7s scores were higher when administered by telephone. Differences by telephone vs face-to-face mode of administration are apparent in cognitive measurement in older adults, and most pronounced for tests of memory and attention that can be easier to answer via telephone. Future investigations are warranted to further evaluate methods to correct for such differences.


Multidisciplinary rounds (MDRs) are scheduled, patient-focused communication mechanisms among multidisciplinary providers in the intensive care unit (ICU).i-Dashboard is a custom-developed visualization dashboard that supports (1) key information retrieval and reorganization, (2) time-series data, and (3) display on large touch screens during MDRs. This study aimed to evaluate the performance, including the efficiency of prerounding data gathering, communication accuracy, and information exchange, and clinical satisfaction of integrating i-Dashboard as a platform to facilitate MDRs.i-Dashboard during MDRs were compared with those of the established electronic medical record (EMR) through direct observation and questionnaire surveys.A cluster-randomized controlled trial was performed in 2 surgical ICUs at a university hospital. Study participants included all multidisciplinary care team members. The performance and clinical satisfaction of i-Dashboard, respectively. For prerounding data gathering, the median time was 10.4 (IQR 9.1-11.8) and 4.6 (IQR 3.5-5.8) minutes using the established EMR and i-Dashboard (P<.001), respectively. During MDRs, data misrepresentations were significantly less frequent with i-Dashboard  than with the established EMR . Further, effective recommendations were significantly more frequent with i-Dashboard than with the established EMR (P<.001). The questionnaire results revealed that participants favored using i-Dashboard in association with the enhancement of care plan development and team participation during MDRs.Between April 26 and July 18, 2021, a total of 78 and 91 MDRs were performed with the established EMR and i-Dashboard increases efficiency in data gathering. Displaying i-Dashboard on large touch screens in MDRs may enhance communication accuracy, information exchange, and clinical satisfaction. The design concepts of i-Dashboard may help develop visualization dashboards that are more applicable for ICU MDRs. ClinicalTrials.gov NCT04845698; https://clinicaltrials.gov/ct2/show/NCT04845698 Philips ICCA is an ICU-specific EMR system that provides information essential for critical care . Patienti-Dashboard. The i-Dashboard (Advantech) was custom-developed under the guidance of multidisciplinary professionals, including physicians, registered nurses, NPs, RTs, pharmacists, and dietitians, rather than only physicians because information for MDRs needs may vary on the basis of the clinical role. Every health care provider working in the surgical ICU participated in developing i-Dashboard. Different professionals held preparatory meetings of their own. Before i-Dashboard was formally implemented for clinical use, the structure and layout were repeatedly revised and tailored to achieve broad acceptance among directors of multidisciplinary providers in the ICU.This study evaluated the performance of the first version of i-Dashboard is summarized in i-Dashboard was designed to substitute as a station whiteboard with lists of patients, information to aid emergency evacuation, and on-duty physicians, nurses, and NPs. Patient-level data in i-Dashboard were modified from the MDR checklist and digitally transformed. Therefore, data were preidentified and retrieved from different origins in the established EMR environment, especially ICCA. Instead of database-centered displays, these data were reorganized on the basis of the requirements of different professionals or disciplines to form dashboard pages  and element blocks. In addition to colored signaling for values outside the reference ranges, i-Dashboard was designed to support built-in automated calculation of severity scores  and visualization of time-series data to expedite navigation of patient condition. Time-series data  that were transformed into line charts can be accessed through the hyperlinks located at the left upper corner of element blocks on the overview page. The pages of time-series data were designed to support both fixed  and relative custom time frames are available.The architecture of i-Dashboard are summarized in i-dashboard. A K8s-based WISE-PaaS 4.0 platform (Advantech) facilitates the integration of diverse devices and communication protocols, making data exchange and system development agile. The entire platform was developed and deployed on 6 VMware servers, each with a 256-GB hard drive and 32-GB memory, and a 24-core Intel Xeon Gold 6248R 3.00 GHz processor. The servers received the data from the Philips ICCA, HIS, and LIS database servers.The technical details underlying i-Dashboard can be accessed through desktop computers with 17- or 22-inch monitors or mobile platforms.In the study units, both the established EMR and i-Dashboard, NPs accessed the established EMR systems, including ICCA, HIS, LIS, and PACS, for data gathering through desktop computers.Prerounding data gathering and MDRs have long been standardized with a structured script for reporting . WithoutMDRs took place outside the patient rooms. To facilitate situation awareness of other participants, NPs delivered the oral case presentation and data communication based on the structured script, including basic information, catheter placement and their duration, vital signs, laboratory data, medications, input or output and nutrition, critical values, major image findings, consultations, and other major events . The inti-Dashboard, we prepared education materials  through the hyperlinks on the overview page. Time-series data can be rearranged on the basis of different time frames as requested. In addition, the RT, pharmacist, and dietitian could take turns operating i-Dashboard and use the RT-pharmacist-dietitian page to demonstrate valuable information of their professionals. Finally, the intensivist used i-Dashboard to facilitate bedside teaching. The goals were documented after consensus was reached.During MDRs, all MDR participants gathered in front of a 55-inch 4K interactive touch screen, approximately 3 m away with clear sightlines. The touch screen allowed users to enter different pages of Two NPs who were not directly involved in MDRs and patient care were trained to audit the processes. The 2 NPs have 8 and 6 years of ICU experience, respectively. The 2 observers were temporarily exempted from clinical work during the study period. To ensure adequate training in the study methodology, personnel piloted data collection and evaluation of communication accuracy were performed by the 2 observers and supervised by the senior investigator (CHL) during a 4-week run-in period. The 2 observers performed in-field observation and audio recordings and audited the process together using a standardized form  to reducPrimary outcomes of interest included time of data gathering before MDRs and communication accuracy during MDRs. The amount of time that NPs spent gathering clinical data before rounding was recorded. Communication accuracy was evaluated on the basis of the items listed on the standardized form  through i-Dashboard. The recommendations initiated by RTs, pharmacists, and dietitians, as exemplified in i-Dashboard as an information management tool was investigated using Likert scale–based questionnaires  minutes. The intraclass correlation coefficient of this pilot study was 0.47. The corresponding estimations were treated as true parameters. Thus, enrolling approximately 144 patients during 24 unit-weeks would provide a power of 90% at a type I error rate of 0.05 to detect an intervention effect of 3 minutes between groups. Categorical variables were analyzed using the chi-square test or Fisher exact test as needed. Continuous variables and the Likert scale were analyzed using the Mann-Whitney U test. Statistical analyses were performed using R software . A 2-tailed P value of <.05 was considered statistically significant.Before the trial began, it was estimated that 12 MDRs would be observed per unit in a 2-week period. To estimate the required sample size, we used a cluster-randomized controlled trial design to account for the positive intraclass correlation expected among members of the same group or cluster. Observational pilot data for prerounding data gathering were collected. The results showed that the mean time difference between the use of the established EMR and i-Dashboard  than with the established EMR environment . In addition, both laboratory and nonlaboratory data misrepresentations were reduced using i-Dashboard compared with the established EMR. Among audited laboratory results, only one misrepresentation (0.2%) occurred among 577 data points with i-Dashboard. In contrast, 163 (32.3%) misrepresentations occurred in 505 data points with the established EMR environment (P<.001), and the majority (95.1%) of these misrepresentations were omissions (155 data points).Regarding communication accuracy during MDRs , data mii-Dashboard than with the established EMR, whereas the proportions of 2 and 3 recommendations were higher in MDRs with i-Dashboard than with the established EMR. The number of effective recommendations was significantly higher in MDRs with i-Dashboard than with the established EMR (P<.001).Regarding information exchange , the proi-Dashboard (response rate=84.4%). Grouping results of responses to Questionnaire 1 in term of task productivity, task innovation, customer satisfaction, and management control are shown in i-Dashboard was superior to the established EMR in task productivity . Further, i-Dashboard was superior to established EMR in task innovation , customer satisfaction , and management control . These findings suggest that i-Dashboard outperformed the established EMR across the 4 dimensions.A total of 76 health care providers . In this study, we estimated a reduction of ~60 mouse clicks per patient using i-Dashboard versus the established EMR before the trial. For experienced NPs, this improvement can be translated to a 6-minute reduction, compatible with what has been observed (5.8 minutes) during the trial. Health care providers can reduce cognitive fatigue in the data extraction process and pay attention to more productive information.Visualization dashboards can inform decision-making and support behavior change in public health and health care services -25. A rei-Dashboard as a visualization aid for MDRs.The ICU is a dynamic environment in which multiple information pathways and personnel interactions facilitate patient care. Specially trained health care professionals in the ICU rely on interdisciplinary communication to make effective clinical decisions. Reliance on a single individual in patient data communication during MDRs represents a coping strategy for an EMR system that does not automatically provide an effective visualization display of the data needed . Consisti-Dashboard, participants of different professions had a space and time interval of their own, thereby enhancing the sense of participation and collaboration. i-Dashboard may help the RT, pharmacist, and dietitian focus on expressing their thoughts explicitly, as other participants could obtain numerical information from the visualization aid. Their inputs were thus more likely to be valued and adopted by the multidisciplinary care team. The positive impacts of i-Dashboard on objective outcomes were corroborated by the increased perception of situation awareness and team participation in participants, as revealed in the questionnaire results.While structured presentation using checklists and explicit definitions of each health care provider’s role may facilitate MDRs, allied health care provider perceptions of not being valued by rounding intensivists have been recognized as an unfavorable factor that impairs the productivity of MDRs. In MDRs with i-Dashboard . A recent study has demonstrated valuable experience regarding the evolution process of an emergency department dashboard, which has undergone several significant revisions to respond to feedback from users [i-Dashboard.We found that after the study period, health care providers in study units started to propose novel ideas that might improve the usability of om users . Currenti-Dashboard on patient outcomes. Further studies are warranted in this respect. Second, the designers of i-Dashboard were part of the team that conducted the study and assessed the outcomes. Successful implementation of dashboards greatly relies on user experience. However, the participation of this paper’s authors in the development process potentially leads to biases in assessing outcomes [i-Dashboard was custom-developed with reference to our established EMR environment. Therefore, it is difficult to extrapolate our study findings directly into ICUs of other hospitals, possibly limiting their generalizability. Nevertheless, visualization dashboards are intended to reduce the time spent on the data gathering process and improve situation awareness and navigation [i-Dashboard obtained in a mature MDR environment suggest that these design concepts may help develop or modify visualization dashboards in the ICU more applicably for MDRs through technological advancements.Our findings must be interpreted within the context of the study limitations. First, traditional study outcomes in critical care, such as mortality and length of stay, were not evaluated in this study. No conclusion can be achieved regarding the effect of outcomes . Finallyvigation ,21. The i-Dashboard as an information management platform for MDRs. The implementation of i-Dashboard can increase efficiency in prerounding data gathering. As a visualization aid, i-Dashboard displayed on large screens enhances communication accuracy and information exchange during MDRs. Establishing care team–designed visualization dashboards as an integrated information platform may reinforce the communication quality of MDRs, thus potentially improving the workflow process in the ICU.In conclusion, we developed


Bone healing is a multifarious process involving mesenchymal stem cells, osteoprogenitor cells, macrophages, osteoblasts and -clasts, and chondrocytes to restore the osseous tissue. Particularly in long bones including the tibia, clavicle, humerus and femur, this process fails in 2–10% of all fractures, with devastating effects for the patient and the healthcare system. Underlying reasons for this failure are manifold, from lack of biomechanical stability to impaired biological host conditions and wound-immanent intricacies. In this review, we describe the cellular components involved in impaired bone healing and how they interfere with the delicately orchestrated processes of bone repair and formation. We subsequently outline and weigh the risk factors for the development of non-unions that have been established in the literature. Therapeutic prospects are illustrated and put into clinical perspective, before the applicability of biomarkers is finally discussed. A broken bone starts to heal at the moment of fracture. The bone tends to re-unite in either a direct or indirect manner through callus formation, and with or without surgical help . Since PAccording to the AO principles, a non-union is a fracture that has not healed after 6 months. This definition is used by most surgeons in daily practice ,9. As deIt should be noted that primary bone healing and secondary bone healing occur in different fracture types. Primary bone healing is achieved by rigid stabilization leading to interfragmentary compression and minimal motion of the fracture (<0.15 mm contact zones) . In consThis review summarizes the epidemiological aspects of fractured bones and the socioeconomic consequences of non-union occurrence. Subsequently, the key cellular players in the (non-)physiological healing process are described and their disturbance briefly discussed. These cellular insights are followed by a systematic review of the risk factors for non-union. Finally, therapeutic options are outlined and the application of potential biomarkers discussed.For the systematic review of the risk factors for non-union, the Medline, Cochrane, and Google Scholar databases were consulted. Original articles, meta-analyses, and reviews were selected and considered. Following keywords were included: “pseudarthrosis”, “non-union”, “delayed healing”, and the Boolean operator “AND” combined with “bone healing” or “fracture” or “fracture healing”. Abstracts from the earliest available records until November 2022 were considered, and reference lists of original articles and reviews were manually evaluated to identify additional relevant studies. The respective algorithm is demonstrated in This study followed the participants, interventions, comparisons, outcomes, and study design (PICOS) framework .The population of interest were humans who had experienced a fracture.The exposure was fracture treatment, either conservatively or operatively, in both the study group and control group.Comparators were considered throughout the review, i.e., smoking status or fracture characteristics.The outcome parameters were the time until complete healing or time until non-union was declared.All studies were eligible, with the exception of narrative syntheses, case studies or studies not available in the English language or as a full text.From 3017 results and after screening all headings, 626 abstracts remained, out of which 23 original articles and 5 reviews were included.Studies were excluded if their manuscripts were not available as full text, as were articles exclusively containing animal studies. Biomechanical studies were also excluded.For the narrative review particularly focusing on the cellular elements contributing to (impaired) fracture healing, additional animal studies were included due to a lack of human data.Non-unions appear rarely, but represent one of the most severe complications in trauma and orthopedic surgery. Their frequency depends on the applied definition and varies between 2–10% of all fractures, but may reach 50% in open tibial fractures ,15,16. DThe highest rates of non-unions were observed in the tibia, clavicle, humerus and femur, and interestingly were not more frequent in older populations ,20.Patients with non-union suffer from tremendous secondary functional deficit and are frequently unable to participate in daily life, and substantially benefit from a structured therapeutic approach taking into account the occurrence of infection, impaired biology, and concurrent metabolic disorders . An effeThe approximate costs for treatment of long-bone non-unions are estimated to be at least USD 11,333 in the US , USD 17,The fracture process can be subdivided into four intergradient stages. The initial stage of fracture hematoma and inflammation is followed by the second stage of cartilage formation with angiogenesis . The third stage is characterized by cartilage removal and calcification , before the fourth stage of chronic bone remodeling concludes the healing . The comUndifferentiated MSCs display a “diamond” component, representing the osteogenic origin of local proliferation in the fracture callus . IntriguAs expected, aging appears to impact the total number of MSCs available at the fracture site and their differentiation potential. While the total number of MSCs harvested was significantly higher in aged mice, the number of colonies formed was substantially lower compared with young animals . In humaThe efficiency of MSC transplantation in the treatment of bone non-union has not been clinically validated. Although four clinical trials have been completed, none have yet published their results . Nonetheless, bone grafting with either autograft or allograft containing MSCs  remains the current gold standard in patients with incomplete bone healing .A cell-free approach to treatment of bone defects involves MSC-derived extracellular vesicles (MSC-EVs) . These mDespite several experimental approaches, the exact mechanisms by which MSCs interfere with the fracture-healing process are poorly understood. In addition, cellular processes and components involved in fracture healing are yet to be elucidated in detail . InconsiMacrophages are myeloid lineage cells, differentiated from hematopoietic stem cells, that reside in periosteal and endosteal tissues and are frequently referred to as osteomacs . M1 macrOsteogenic cells arise from the local periosteum and potentially to a small proportion from C-X-C chemokine receptor type 4 (CXCR4)-positive circulating osteoprogenitors . In a smA decline of progenitor cells in bone marrow aspirates with age has been demonstrated in women, but not in men . InteresWhile NOTCH signaling is dispensable in both osteoblasts and chondrocytes, it is imperative in osteoprogenitors to enable normal bone healing .Osteoblasts are bone-synthesizing cells originating from MSCs. They appear to be particularly important in the middle stages of bone healing. Osteoblasts have been found to be unaltered in the early stages of non-union development, while in observations of the osteoblastic maturation process after 5 and 10 weeks, runt-related transcription factor 2 (RUNX2)- and osteocalcin (OCN)-positive cells were significantly downregulated in a murine atrophic non-union model . In addiOsteocytes are mechanosensitive cells that respond to the deformation of surrounding tissue via flow-induced shear stress on their surfaces. Their exact function and the role of the lacuno-canalicular network has been outlined in detail elsewhere . These mOsteoclast activity was markedly increased in human scaphoid non-unions, and expanded osteoclastogenesis may be one factor leading to non-union . While oChondrocytes are hypothesized to transform into osteoblasts, as demonstrated in Agc1CreERT::Ai9 mice, in which SRY (sex determining region Y)-box 2 (SOX2), octamer-binding transcription factor 4 (OCT4), and NANOG were co-stained in chondrocyte-stem-cell-like cells. This mechanism may be perturbed in non-unions ,72. In fThe importance of vascularization for the healing fracture has been highlighted in several studies ,75,76. IIn hypertrophic non-unions, the density of blood vessels was comparable to that in regular healing bone in a human study of non-union vs. healed fractures, in scaphoid non-unions, and in animal models ,77,78. WOverall, the roles of several key cellular players within the healing bone have been well studied. However, the mechanistic details of non-union, its cellular origins, and related disturbances remain largely unknown to date. With novel techniques such as single-cell RNA sequencing (scRNA-Seq) and the broad applicability of spatial transcriptomics, major progress is to be expected in the field of bone (non-)healing in the coming decades ,80,81.Numerous studies have analyzed risk factors for bone non-union. The following systematic review see also  evaluateAge itself may present a risk factor for non-unions in the clavicle ,84,86, hA larger systematic meta-analysis concluded sex not to be a risk factor for non-union (odds ratio [OR]: 1.0) .Smoking has been validated as a risk factor for non-union in the clavicle , diaphysOpen fractures have been associated with higher rates of non-union . In a syDisplacement has been analyzed in fractures of the clavicle. Here, displaced fractures are a predictive factor for non-union . In a prDiabetes appears to be a risk factor for non-union, as shown in a meta-analysis . HoweverObesity has been identified as a risk factor for non-union even in pediatric patients ,104, andWhen osteoporosis is included in the list of risk factors for non-union, evidence is conflicting. Despite some evidence of osteoporosis  negatively affecting bone union in according to univariate analysis , a prospLiterature on the consumption of alcohol and its effect on fracture healing is inconsistent. In a prospective trial involving 122 patients with femoral neck fractures, alcohol was a risk factor according to a multivariate analysis , althougDrug treatment may also intervene with physiological bone healing. The most prominent drugs in this respect are NSAIDs and steroids. NSAIDs have been frequently discussed as a risk factor for non-union. In a large matched-control study, administration of NSAIDs 12 months before fracture led to an OR of 2.6 for fracture non-union , while a+, CD8+, and regulatory T cells [Immune dysregulation has been hypothesized to impair fracture healing. When the initial inflammatory phase and local immunosuppression are efficiently resolved, later stages require immune cells to guide MSC differentiation and activity . In all  T cells . Accordi T cells . The com T cells .To treat bone non-unions, classical principles  and novel approaches  can be applied.The administration of BMP-2, BMP-7, platelet-rich plasma (PRP), and MSCs has been demonstrated in a recent meta-analysis to reduce time taken for bone repair . As an eAnother method for the treatment of delayed healing or non-unions is low-intensity pulsed ultrasound (LIPUS), which in 1994 was approved by the FDA for acceleration of bone healing . DespitePulsed electromagnetic field stimulation (PEMFs) represents another approach for the treatment of bone non-unions, and is FDA-approved ,131. PreBiomaterial-supported therapies for bone regeneration include various different approaches. Among these, bone-graft substitutes including a combination of bio-active molecules and cell-based supplements with osteoprogenitors have been applied in vivo . Bone-grThe latter approach also represents a cell-free alternative. MSC-derived EVs alone have been shown to improve fracture healing in rodent models ,46.The convergence of mechanical and biological considerations, in addition to adequate vascular supply at the healing site and the physiological host state in the “diamond concept”, conceptualizes a framework of fracture repair comparable to a polychemotherapy attack on non-unions. Within this context, the healing process is facilitated by osteoinductive mediators (cytokines released by macrophages), an underlying matrix (extracellular matrix promoting “homing” and migration of osteogenic cells), osteogenic cells , and mechanical stability (sensed by osteocytes) ,144. In TGF-β1 has been described as a potential biomarker for non-union since a decline was detected in patients with diaphyseal long bone fractures , which wAlthough serum of non-union patients has been shown to impair bone marrow (BM)-MSC proliferation in vitro, the underlying factors were not identified, and cytokine levels were found to be similar in non-union and union serum samples .However, pre-clinical studies have been promising. In a rat critical size defect model, cytokines including IL-10 and myeloid-derived suppressor cells (MDSCs) were significantly elevated in blood one week after treatment, prior to poor bone healing outcomes. Interestingly, blood B-cell levels were positively correlated to bone volume as early as one week after treatment, indicating an early predictor for successful healing. Given the importance of B-cell maturation for bone healing, these cells might be promising markers for bone repair, although clinical trials remain lacking . MoreoveIn a small study , Annexin A3 (ANXA3) was found to be significantly upregulated in blood samples of patients with non-unions compared with fracture unions . As indiThese findings indicate that no reliable biomarker for non-union has been identified that is sufficiently understood and clinically confirmed.Bone non-union occurs in 2–10% of all fractures. Although the cellular components of bone healing are partially understood, the mechanisms by which bone fails to heal are manifold and remain largely unexplored. Among the main risk factors for non-union are smoking, open fractures, and certain medications including NSAIDs. However, the literature lacks prospective randomized controlled trials to verify the results of an abundance of retrospective studies. The diamond concept has proven itself over the years, providing a six-sided convergence of considerations leading to non-union, and offering a guide to its treatment. Unfortunately, reliable biomarkers for non-union are scarce. Non-union remains a pivotal challenge for trauma surgery and orthopedics, and uncovering the underlying pathological mechanisms of non-union in order to develop new therapeutic options represents a major challenge for researchers and surgeons in the 21st century.