In this article, we describe the modulation of pathophysiology by SSO2, review the existing trial data and present our first impressions with the technique in real clinical practice.Optimal medical therapy for secondary prevention following acute myocardial infarction reduces non-fatal ischaemic events. Intensive antithrombotic or lipid-lowering approaches have failed to significantly lower mortality. In the past, reduction of infarct size in patients undergoing primary percutaneous revascularisation for acute myocardial infarction had been considered as a surrogate outcome marker. However, infarct size measured by magnetic resonance imaging or SPECT is strongly associated with all-cause mortality and hospitalization for heart failure within the first year after an acute myocardial infarction. Intracoronary administration of super-saturated oxygen (SSO Acute myocardial infarction (MI) is one of the major contributors to cardiovascular morbidity and mortality. In the acute phase of ST-elevation MI (STEMI), ventricular arrhythmias and cardiogenic shock are the main drivers of mortality contributing to in-hospital mortality of approximately 10% and time from first medical contact to primary percutaneous coronary intervention (PCI) is a strong predictor of an adverse outcome ,5,6,7,8.A meta-analysis of 10 randomized primary PCI trials including more than 2500 STEMI patients showed that infarct size measured within 1 month by cardiac magnetic resonance (CMR) imaging or technetium-99m sestamibi single-photon emission computed tomography (SPECT) was strongly associated with all-cause mortality and hospitalization for heart failure within 1 year . A centrOne of the approaches to reduce reperfusion injury was infusion of the mitochondria-targeted peptide, Bendavia, to reduce mitochondria-produced reactive oxygen species, which are believed to be centrally involved in reperfusion injury. While this approach seemed to reduce infarct size in animal models , no effe2 at 4 atmospheres [As local hypoxia was considered as a driving force for free-radical generation and mitochondrial dysfunction, the first experimental step to counteract this was hyperbaric oxygen therapy (HBO). In the 1950s, initial experiments of coronary sinus occlusion leading to higher coronary sinus pressure resulted in reduced mortality in animal models of coronary arterial occlusion . Hence, ospheres . Similarospheres . These eospheres . In 1973In 1993 Sterling et al. started to investigate in a rabbit model whether HBO affects infarct size. They surgically occluded one branch of the left coronary artery for 30 min prior to reperfusion. In their experimental setup they observed significant infarct size reduction from 42 to 10% when both ischaemia as well as reperfusion occurred during hyperbaric conditions and to 15% when only reperfusion was hyperbaric . At the Placing patients with acute STEMI into HBO chambers might pose several logistical problems including poor accessibility, difficulties for monitoring, claustrophobia and necessary decompression phases when performing the treatment for multiple hours. Blood hyperoxygenation might be possible using conventional extracorporeal membrane oxygenation (ECMO). However, this approach is limited by invasiveness, activation of coagulation and inflammation and the need for specialized personal for monitoring. It would counteract the minimal-invasive approach using radial access for prevention of bleeding when performing primary PCI nowadays. A solution for all those problems would enable easy hyper-oxygenation of blood components able to reach the peripheral myocardium. Aqueous oxygen has an oxygen-carrying capacity which is approximately 10-fold greater than those of blood. The advantage of aqueous oxygen is that it can be infused into other aqueous media at normal pressure without formation of gas bubbles . Followi® system  produces aqueous oxygen from bottled oxygen for medical use and sterile sodium-chloride solution. It requires arterial blood either from a singular femoral 4F-sheath in case of trans-radial catheterization or escalation of femoral access to a 7F-sheath which can host both arterial drainage as well as the guiding 5F-catheter for coronary reinfusion of hyperoxaemic blood. For easy handling in the cath-lab, a single-use sterile cartridge is placed in the TherOx® system and connected with a saline infusion as well as one line for drawing arterial normoxic blood and one line to connect with the coronary guiding catheter placed in the left-main coronary artery. Within the cartridge, aqueous oxygen solution was produced and injected into the circulating blood. The guiding catheter then delivers hyperoxaemic blood to the coronary system [The TherOxy system .2 appears to be controversial at first sight. However, animal experiments suggest that aqueous oxygen does not result in an increased formation of reactive oxygen species but rather reduces them in an animal setting [The proof of principal for aqueous oxygen therapy was provided by Spears et al. in an animal model using dogs subjected to coronary hypoxaemia. In that model, intra-arterial aqueous oxygen preserved left-ventricular ejection fraction . In a pi setting .2 provides hyperoxemic levels of oxygen, which are up to 10 times higher than normal, of dissolved oxygen when it is infused in the infarct-related artery. This allows for high oxygen diffusion before flow is restored downstream. Increased oxygen delivery via the plasma to endothelial cells can prevent severe cell edema, allow better perfusion and correct persistent myocardial ischemia, particularly in regions where erythrocyte flow is impaired by compromise of the microvascular luminal integrity [2 in contrast to LV unloading or hypothermia either requires or benefits from revascularisation prior to application. On the other hand, this provides the clinical advantage of not delaying primary PCI compared to the other methods [Experimental data support the pathophysiological hypothesis that microvascular ischemia caused by microvascular obstruction contributes to myocardial tissue injury over a prolonged time during reperfusion. It is believed that SSOntegrity . As the  methods ,22.2 concept and preclinical studies to clinical studies finally leading to FDA approval [After the encouraging results from animal experiments, first smaller pilot trials were started in humans with acute MI. A recent review extensively describes the evolvement from hypothesis generation over introduction of the SSOapproval .® device was reported in 2002 by an American–Italian multicentre investigation. In this pilot trial, 29 patients with acute MI undergoing primary PCI received hyperoxaemic blood infusion into the infarct-related artery for 60–90 min after successful revascularisation. Hyperoxemic reperfusion was well tolerated by all patients and neither clinical, electrical nor hemodynamic instability was observed during the aqueous oxygen infusion. The primary purpose of the study was to evaluate the feasibility and safety of performing hyperoxemic reperfusion in patients undergoing primary PCI for acute MI. While safety could be demonstrated, it remained unclear whether there was a potential benefit regarding reduction of infarct size or improvement in left-ventricular function [Initial experience with aqueous oxygen in humans using the TherOxfunction .An Italian study reported 27 patients with acute anterior AMI treated with primary PCI within 12 h after symptom onset receiving selective hyperoxemic perfusion into the left anterior descending coronary artery for 90 min. They were compared to 24 anterior STEMI patients matched in clinical and angiographic characteristics and treated with conventional primary PCI. Recanalization and hyperoxemic perfusion were successfully performed in all patients. After hyperoxemic perfusion patients showed shorter time-to-peak creatine kinase release and a higher rate of complete ST-segment resolution. A significant improvement of mean LV ejection fraction from 44 to 55% was observed after 3 months in hyperoxemic perfusion patients only. Therefore, the authors concluded that hyperoxemic perfusion using the aqueous oxygen technique is associated with LV function recovery .The first prospective, randomized trial of intracoronary hyperoxemic reperfusion after primary PCI was the Acute Myocardial Infarction With Hyperoxemic Therapy (AMIHOT I) trial published in 2007 . In AMIH2 and 79 were controls. The analysis was planned to include all patients from both groups in AMIHOT I meeting the AMIHOT II inclusion criteria for a pooled analysis, which finally included 124 control and 258 SSO2 therapy patients. There was no significant difference in MACE rate, neither in AMIHOT II alone nor in the pooled group. Regarding non-MACE adverse events, the rate of hemorrhagic adverse events was higher with SSO2 than in controls  predominantly attributable to access site related bleeding. However, at that time 9F sheaths were used in 53%, 8F sheaths in 22%, and 7F sheaths in 25% of patients. During the trial, the type of catheter used for SSO2 therapy did change accompanied by preferring a single unilateral instead of dual bilateral femoral artery access. This resulted in less access site bleeding complications on SSO2 during the later phase of the trial compared to the initial phase. On the ischemic site of events, the rate of stent thrombosis trended to be higher in the SSO2 group , but it has to be acknowledged that in AMIHOT II patients received early-generation drug-eluting stents  and the P2Y12 inhibitor used had been clopidogrel [2 therapy reduced infarct size determined after 14 days by 99mTc-sestamibi SPECT from 26.5–20.0% (adjusted p = 0.03). In the pre-planned pooled analysis form both AMIHOT-trials, infarct size was reduced from 25.0–18.5% (p = 0.02). Among 154 patients with a baseline LV-EF below 40%, SSO2 therapy reduced infarct size from 33.5–23.5%. Therefore, the overall conclusion from both AMIHOT trials was that intracoronary SSO2 infusion into the infarct-related artery following successful primary PCI is feasible and safe and reduces myocardial infarct size in patients with acute anterior AMI presenting within 6 h of symptom onset [2 therapy, this trial indicates that patients with apparent ventricular compromise (LV-EF < 40%) suggestive for larger infarct zone are the ones profiting most from the intervention.Based on the positive findings in the anterior AMI group treated within 6 h in AMIHOT I, a second prospective, multicentre trial (AMIHOT II) was conducted only randomizing patients with anterior AMI and symptom onset < 6 h prior to primary PCI and reported in 2009. Of 301 patients randomized in the intention-to-treat analysis, 222 received SSOpidogrel . In AMIHom onset . In addi2 directly via a guiding catheter in the left-main coronary artery evolved. Therefore, the IntraCoronary Hyperoxemic Oxygen Therapy (IC-HOT) trials was conducted. IC-HOT, published in 2019, was a single-arm, safety trial including 100 patients with anterior STEMI presenting within 6 h of symptom onset [2 delivery was successful in 98% of patients. NACE rate at 30 days was 7.1%. Infarct size was 24% at 4 days and 19% at 30 days. Therefore, the conclusion was that infusion of SSO2 via the left-main coronary artery was feasible and safe [To overcome the limitation of selective infusion catheters placed within the proximal segment of the infarct-related coronary artery, the idea of applying SSOom onset . The priand safe .The clinical findings observed in the three studies are summarized in ® system for application of SSO2 therapy in everyday clinical routine. In terms of practicability during acute STEMI treatment in the cath-lab, the SSO2 technique has one major advantage over other approaches aiming for infarct size reduction in large anterior MIs such as LV-unloading by Impella [2 therapy. While the assigned operator performed routine primary PCI, the core team set up the TherOx® system. After initial training, it was easily possible to exchange the femoral sheath in case of primary femoral access or to place the additional femoral 4F-sheath in case of a primary radial approach. The perfusion catheter, which is a 5Fr coronary diagnostic catheter, is rapidly placed in the left main coronary artery, while the TherOx® system is primed with blood, and SSO2 therapy can start within 4 min after the last angiography at the end of primary PCI. The patients tolerated the 60 min of SSO2 administration well. The downside in a busy cath-lab might be that the patients stay on the cath-lab table for one hour of SSO2 therapy.At Hannover Medical School, in March 2021 we had the opportunity to be the first in Europe to gain experience with the commercially available TherOx Impella  or thera Impella : the add2 in anterior AMI patients, all our patients undergo distinct imaging for LV function and detection of myocardial inflammation and necrosis using transthoracic echocardiography, cardiac magnetic resonance imaging and 99mTc- tetrofosmin perfusion SPECT [2 therapy. In the meantime, German authorities have demanded that prior to reimbursement a large randomized controlled trial with patient-relevant outcome measures has to be conducted to prove the additive benefit suggested by the previous smaller clinical trials.To evaluate further the potentially beneficial effect of SSOon SPECT ,48. Once2 therapy is easy to set up and applicable during standard clinical treatment of acute AMI patients by primary PCI. The application does not delay standard procedures prior to revascularization and can start within minutes after PCI.Overall, SSO2 therapy using aqueous oxygen demonstrated proof of principle in animals and showed feasibility and safety in humans undergoing primary PCI for treatment of acute anterior STEMI. While larger clinical trials powered to demonstrate reduction of major adverse cardiovascular events directly are still lacking, evidence from almost 360 patients indicates a potential to reduce infarct size at least in anterior STEMI receiving treatment within 6 h of symptom onset. Aggregate data from AMIHOT I, AMIHOT II, and IC-HOT suggest that SSO2 therapy might reduce infarct size in those patients from approximately 25 to 27% of the LV to about 19% of the LV [2 therapy, would reflect an approximate 17% reduction in all-cause mortality and heart failure hospitalization within 1 year. Considering 1-year mortality or heart failure hospitalization rates in these patients of 2–3% each, an adequately powered randomized controlled outcome trial would have to be unfeasibly large and, therefore, difficult or even impossible to conduct. Using infarct size in early presenting anterior STEMI based on the effects of the older trials, an adequately powered, randomized-controlled trial is feasible with approximately 200 patients per group.SSOf the LV ,45,46. N2 therapy. The earlier trials showed some development from trial to trial. AMIHOT I identified the anterior MI presenting within 6 h of symptom onset as the subgroup profiting most from the procedure [2 therapy had larger infarct size (33.5% vs. 16.5% of LV) than those with preserved LV-EF and SSO2 reduced infarct size by an absolute 10% [2 could translate into a clinical benefit of almost 36% regarding reduction of mortality and heart failure hospitalization within 1 year [2 therapy while avoiding selective catheterization of the left-anterior descending coronary artery by simply reperfusing hyperoxemic blood through a guiding catheter into the left-main coronary artery, making the procedure extremely easy and rapid to conduct [It will be important to identify patients who are ideally suited to profit from SSOrocedure . AMIHOT lute 10% . Identifn 1 year . Finally conduct .2 therapy is the least invasive one. Using Impella devices for LV unloading starting prior to and delaying revascularization requires a femoral 14F access [In comparison to other approaches aiming for infarct size reduction in AMI, which almost all focus on large anterior STEMI within 6 h of symptom onset, SSOF access . PerformF access . FurtherF access .2 through cooled blood providing additive intracoronary hypothermia [In the future, it might be interesting to see whether a combination of different approaches reducing infarct size in addition to primary PCI will provide additive benefit to the patient. However, this will require that the individual interventions provide a benefit on their own. Respective clinical trials are either on their way or in the stage of planning. Theoretically, it is appealing to think about combining these strategies, for example, by infusing SSOothermia  or applyothermia .2 therapy in acute STEMI is feasible and safe. It does not delay state-of-the art revascularization and can start within 5 min after the end of primary PCI. Earlier studies demonstrated an approximate absolute 6–7% reduction of infarct size in anterior STEMI, which translates into almost 20% reduction of 1-year all-cause mortality and heart failure hospitalization. However, an adequately powered clinical trial under contemporary PCI conditions is needed.In summary, intracoronary application of SSO


Longer time to LTC (in weeks) was significantly associated with shorter OS in univariate analysis (p = 0.04), but not in an adjusted model (p = 0.14). Both univariate and adjusted analyses showed that longer time to reach LTC was significantly associated with shorter PFS . Conclusions: These findings show that the longer time to LTC is not an independent predictor of OS, but suggest that PFS may be significantly shorter in patients with longer time to LTC.Background: Whether the number of loco-regional treatment sessions and the time required to obtain local tumor control (LTC) affects the prognosis of patients with hepatocellular carcinoma (HCC) remains controversial. This study aimed to determine whether a longer time to LTC is a significant and independent predictor of poor treatment outcomes. Methods: In this retrospective study, we analyzed data of 139 treatment-naive patients with HCC who were not eligible for a treatment other than transarterial chemoembolization (TACE) at baseline. The outcome analyses were performed using the Cox proportional hazard model and Kaplan–Meier method, while the overall survival (OS) and progression free survival (PFS) were the primary study endpoints. Results: Overall, LTC was achieved in 82 (59%) of patients, including 67 (81%) patients who achieved LTC following TACE sessions alone and 15 (19%) subjects required additional ablation session. The median OS did not differ significantly between groups that needed 2, 3, or >3 locoregional treatment sessions to achieve LTC ( Therapy for hepatocellular carcinoma (HCC) consists of various treatment modalities, including liver transplantation, resection, systemic treatment, and locoregional therapies ,2. DespiPrevious studies that examined the association between early LTC and long-term clinical outcomes have shown conflicting results ,10,11,12This study aims to determine whether a longer time to LTC is a significant and independent predictor of poor treatment outcomes. For this purpose, we analyzed data for treatment-naive patients with HCC who achieved LTC after repeated locoregional sessions.In this retrospective study, we analyzed consecutive treatment-naive patients with HCC confirmed by the imaging criteria by the American Association for the Study of Liver Diseases who underwent initially at least two conventional TACE sessions between 2016 and 2018. The inclusion criteria were as follows: (1) treatment-naïve patients with at least one HCC lesion, (2) patients were not eligible for other than TACE treatment at baseline, (3) patients had Barcelona Clinic Liver Cancer (BCLC) stage A/B disease, (4) patients had Child–Pugh score (CPS) of A or B, (5) patients had an available dynamic contrast-enhanced CT or MRI liver examinations. The exclusion criteria were: (1) patients that did not complete at least two TACE sessions, (2) other than TACE or ablation HCC-specific invasive treatment , (3) other malignant neoplasm or uncontrolled disease during follow-up, and (4) incomplete clinical data. The final study population and a summary of the enrollment criteria are shown in All patients underwent at least two conventional TACE procedures. During the procedure, 20–40 mL of a 1:1 mixture of lipiodol and doxorubicin was selectively injected into the tumor feeding artery until flow stasis was achieved, with subsequent embolization with gelatin sponge particles . A standard treatment cycle consisted of at least two sessions of TACE at 4–6-week intervals with subsequent imaging (30–90 days after second (or more if indicated) TACE). If a viable tumor was evident, an additional TACE or ablation procedure was performed according to “treatment stage migration” strategy ,18. PatiOverall, 29 patients needed at least 1 percutaneous radiofrequency ablation (RFA) or microwave ablation (MVA) under general anesthesia following initial TACE sessions. The type of ablation needle was selected depending on the target size of the lesion, and then ablation was performed for 4–10 min. Subsequent ultrasound or CT examination was performed immediately after the ablation to exclude active bleeding or damage to adjacent organ.Follow-up examinations were performed by CT or MRI according to the LI-RADS 2018 technical recommendations, and treatment responses were assessed using the LI-RADS treatment response (LR-TR) algorithm . LTC wasBaseline demographic data, laboratory tests, liver function ), and tumor (BCLC) stage were analyzed. The date of the first TACE session was set as an index day. The date of LTC was specified as the date of the last TACE (or ablation) procedure before response evaluation. If a LTC was achieved, patients were followed up by repeated imaging and measurements of serum α-fetoprotein concentration until HCC recurrence was confirmed. The date of death or last clinical follow-up was defined as the end of the follow-up period. Overall survival (OS) was the primary study endpoint. In patients who achieved LTC, PFS was defined as the interval between reaching LTC and the date of reported progression (including detection of new intrahepatic HCC lesions).p  ≤  0.2 derived from univariate regression analyses. SAS software  was used for all statistical analyses and artwork.Kruskal–Wallis and Fisher’s exact tests were used to compare differences between the studied subgroups. Kaplan–Meier method and Log-rank tests with Šidák multiple comparison correction were used to compare survival curves between study subgroups. Univariate and multivariate survival analyses were performed using Cox proportional hazards models. The hazard radios (HRs) in Cox regression models were adjusted for predictors with p < 0.001) (p = 0.93).Final analyses included 139 patients, 82 (59%) of whom achieved LTC. Overall, 67 (81%) patients achieved LTC following TACE sessions alone, while 15 (19%) patients required additional ablation treatment following initial TACE sessions. Subjects who achieved LTC had less advanced BCLC stage, had with fewer treated tumor lesions of a smaller dimension, had a lower percentage of patients with baseline alpha-fetoprotein (AFP) concentration below 200 ng/mL and lower baseline AST level . Patient< 0.001) . The medp = 0.17).In a subgroup of 57 patients with no LTC, locoregional treatment was discontinued after 2, 3, and >3 sessions in 25 (49%), 18 (27%), and 14 (24%) patients, respectively. In those patients locoregional treatments was discontinued due to: untreatable tumor progression in 37 patients (65%) with subsequent palliative or sorafenib treatment ; portal vein thrombosis in 10 patients (17%); decompensation of liver function in 6 patients (11%); other reasons (7%). The median OS was not significantly associated with the number of TACE sessions in patients with no LTC  for three sessions and 1.44  for more than three versus two sessions. Among those who achieved LTC, 42 achieved complete remission after two courses of TACE, whereas 26 required three sessions and 14 required more than three sessions of locoregional treatment. LTC was achieved by 14 (50%) of the 28 patients who received more than three sessions of locoregional therapy. The median OS was 53 months for patients with LTC requiring two sessions of TACE, 60 months for those requiring three sessions, and 39 months for those requiring more than three sessions , while 48 patients (58%) had confirmed progression and 34 individuals (42%) were censored during follow-up period. The KM survival analysis showed that the median PFS did not differ between the LTC groups . A median of 10 weeks (range 5– to 52) between 1st treatment session and LTC was observed. Hazard ratios for the prediction of OS and PFS for prognostic factors generated from the univariate analysis are shown in p = 0.048).The univariate Cox regression model exploring prognostic factors for PFS is shown in Liver transplantation and liver resection are the most efficient therapeutic alternatives for both HCC recurrence and patients survival, thus should be offer to all eligible subjects . HoweverIt has become increasingly clear that post-treatment data may provide important prognostic information that can significantly refine risk stratification in patients with HCC treated with TACE ,21. ThisPrevious findings on the relevance of an early response have been inconsistent and contradictory. Several studies demonstrated that an initial favorable response is a robust predictor of survival following TACE in patients with HCC. For example, Kim et al. showed that the initial, as well as the best overall response, are reliable OS predictors while achievement of early tumor control was the most effective predictor for favorable treatment outcomes . AnotherOverall, a significant proportion of patients did not respond well to the initial TACE sessions , keeping with the study’s results by Georgiades et al. . Of noteThe median OS of patients with BCLC B stage in our cohort was 22 month after initiation of treatment, which is in line with previous reports, including the study by Biolato et al. in which the OS of the entire cohort of patients with stage B BCLC was 23 months . A studyOur work clearly has some limitations. First, it was not possible to analyze the cause of death for all patients with LTC in our cohort. Overall, more than half of LTC subjects had confirmed progression, while the rest were lost during follow-up period. Second, only treatment-naive patients without extrahepatic disease and baseline portal vein thrombosis were included in the analysis. Moreover, patients who underwent ablation before TACE, resection or liver transplantation were excluded from the analysis. Such exclusion criteria increase the homogeneity of the study group; however, this may limit the generalizability of our findings, as HCC treatment is usually multimodal, and the outcomes of patients treated are heterogeneous. Importantly, future efforts to improve patient outcomes should focus on changing modifiable risk factors and identifying patients who may benefit from treatment stage migration strategy ,34. Of nThe results of this study indicate that the time between treatment initiation and achieving LTC adds no prognostic information for the OS of patients with HCC who are not eligible for treatment other than TACE at baseline. However, the evidence from this study suggests that PFS may be significantly shorter in patients with longer time to LTC and there is a need for further analysis to clarify whether these patients may benefit from closer clinical follow-up.


Translocator protein 18-kDa (TSPO) imaging with positron emission tomography (PET) is widely used in research studies of brain diseases that have a neuro-immune component. Quantification of TSPO PET images, however, is associated with several challenges, such as the lack of a reference region, a genetic polymorphism affecting the affinity of the ligand for TSPO, and a strong TSPO signal in the endothelium of the brain vessels. These challenges have created an ongoing debate in the field about which type of quantification is most useful and whether there is an appropriate simplified model.This review focuses on the quantification of TSPO radioligands in the human brain. The various methods of quantification are summarized, including the gold standard of compartmental modeling with metabolite-corrected input function as well as various alternative models and non-invasive approaches. Their advantages and drawbacks are critically assessed.Researchers employing quantification methods for TSPO should understand the advantages and limitations associated with each method. Suggestions are given to help researchers choose between these viable alternative methods. For TSPO, it was first employed in 1C-(R)-PKK1–k4) and macro-parameters that are made up of a combination of these constants -PBR28, known as 2TCM-1K -DPA714 in healthy volunteers and reported that 2TCM-1K fitted the data better than conventional 2TCM; they also found that TSPO mRNA levels strongly correlated not only with Kb but also with mRNA markers of endothelial cells [11C]-(R)-PK11195 had the lowest affinity, [18F]-DPA714 had middle affinity, and [11C]-PBR28 had the highest affinity. They found that models that accounted for vascular binding were preferred for all three ligands using the parsimony criteria [Kb parameter was related to ligand affinity -PK11195 < [18F]-DPA714 < [11C]-PBR28). Finally, a study using [11C]-PBR28 and partial pharmacological blockade showed that 2TCM-1K described the data better than the 2TCM in two cohorts of young and old healthy volunteers, respectively. This model choice was supported by ex vivo evidence of endothelial TSPO, which showed that TSPO-positive vessels accounted for 30% of vascular volume in cortical and white matter -DPA714 [18F]-PBR111, comparable to VND values estimated using the 2TCM [11C]-ER176 yielded poor results, likely because the VT of MABs is only slightly smaller than that of HABs -DPA714  as well the 2TCM . However of HABs , 43. To VND at the individual level. VND is obtained using data from multiple regions and coupling parameters across all regions. The SIME approach first assumes that VND is uniform throughout the brain, which is a standard assumption in modeling PET neuroimaging data . In the operational 2TCM equation, SIME replaces K1 with k2VND. Optimization of the cost function is then performed simultaneously on all the brain regions, in contrast to conventional independent optimization for each distinct brain region. Because VND is assumed to be uniform across regions, this parameter coupling reduces the number of estimated parameters from 4r to 3r + 1 , improving micro-parameter estimates enough to reliably estimate both VND and VS -PBR28 -PBR28 scans acquired after pharmacological blockade with the selective TSPO agonist XBD173 confirmed good agreement in VND estimation with conventional analysis approaches, although data were acquired only in HABs -PBR28 to analyze data from Alzheimer’s disease patients and age-matched controls, VND values were found to be comparable between groups, and effect sizes calculated with BPND were larger than effect sizes calculated from conventional VT estimates -PBR28 . Simulatiability . Human s in HABs , 46. Intstimates . Althougith SIME .The main practical obstacle to performing full quantitative PET studies is the need for an AIF. Although the rate of serious complications is exceedingly small in expert hands , placing18F]-FEPPA, obtained with a modified independent component analysis algorithm [11C]-PBR28 brain scans from the carotid artery. The IDIFs remained very inaccurate even after scaling with blood samples, probably due to the difficulties associated with estimating the rapid peak and low concentrations of parent radioligand after the peak from the noisy voxels.IDIF obtains the input function directly from a blood pool visible on PET images acquired dynamically. Even without considering the challenges posed by correcting partial volume effects in small brain vessels , the mailgorithm . Howeverlgorithm  used a h18F]-FEPPA and found that PBIF increased the variability of the measurements without reducing invasiveness -PBR28 -PBR28  and has K1/k2 should be the same for the target and the reference region). However, because TSPO is ubiquitously expressed in the brain, a proper reference region does not exist -PBR28 antagonist XBD173 in healthy volunteers, Owen and colleagues found that about half of the VT in the cerebellum was attributable to specific binding. Another study of 35 healthy volunteers found increased binding of [11C]-(R)-PK11195 associated with aging in cortical and sub-cortical regions as well as in the cerebellum -(R)-PK11195 BPND values from the 2TCM using plasma input function were much higher than pseudo BPND values from the SRTM with cerebellum as the pseudo-reference region. In addition, signal in the cerebellum may, in some cases, be higher than that in other regions -PBR28, specifically in subjects with chronic low back pain, amyotrophic lateral sclerosis -(R)-PK11195 and is generally preferred to the standard SRTM because it is better able to discriminate between groups -(R)-PK11195 images. More recently, a supervised clustering approach (SVCA) was proposed by Turkheimer and colleagues [11C]-(R)-PK11195 , all of whom were healthy volunteers [11C]-(R)-PK11195 using three different PET cameras and found that, although SRTM with the whole cerebellum as reference region underestimated BPND compared to SVCA-SRTM, the SVCA-derived curve still seemed to be contaminated by specific binding. Similarly, Zanotti-Fregonara and colleagues [11C]-PBR28 -PBR28  and foun11C]-(R)-PK11195 and [18F]-DPA714, were more separable using SVCA than higher affinity, second-generation TSPO ligands such as [11C]-PBR28 [Rizzo and colleagues  then invC]-PBR28 .fP is the fraction of radioligand in plasma that is not bound to plasma proteins at equilibrium -PBR28 study in patients with alcohol use disorder and healthy controls that used VT/fP as an outcome measure reported that the rs6971 genotype alleles were not significantly different due to the high uncertainty introduced with this measure -(R)-PK11195 PET studies. More extensive validation is warranted for radioligands with higher specific binding, especially by replicating clinical protocols that include both healthy volunteers and patients and where images have been quantified with full kinetic modeling.SVCA should be used preferentially for [The use of compartmental models with an additional vascular compartment for TSPO PET has strengths and weaknesses that need to be considered. Adding a vascular compartment allows differentiation of changes in the outcome parameters due to vascular binding and parenchymal uptake, but these advantages occur at the price of less robust identification of the individual parameters.VND  may result in improved parameter estimation compared to conventional quantification methods, discrepancies across studies suggest that further investigation and validation are needed.Although methods for estimating VT for the fp increases the variability of measurements. Therefore, this correction should preferably be used when a variation in fp is expected, such as when a blocking agent is given, as well as when statistical comparison between two groups shows significant average difference in fp.Correcting From this review of the methods presented and the validation of the different methods, the following suggestions for quantification of TSPO PET studies are made:


Pyrenophora tritici-repentis (Ptr) causes tan (syn. yellow) spot of wheat and accounts for significant yield losses worldwide. Understanding the molecular mechanisms of this economically important crop disease is crucial to counteract the yield and quality losses of wheat globally. Substantial progress has been made to comprehend the race structure of this phytopathogen based on its production of necrotrophic effectors and genomic resources of Ptr. However, one limitation for studying Ptr in a laboratory environment is the difficulty to isolate high spore numbers from vegetative growth with mycelial contamination common. These limitations reduce the experimental tractability of Ptr.The necrotrophic fungal pathogen Ptr that yields robust, high quality and pure spores. Our methodology encompasses simple and reproducible plugging and harvesting techniques, resulting in spore yields up to 1500 fold more than the current sporulation methods and was tested on multiple isolates and races of Ptr as well as an additional seven modern Australian Ptr isolates. Moreover, this method also increased purity and spore harvest numbers for two closely related fungal pathogens (Pyrenophora teres f. maculata and f. teres) that cause net blotch diseases in barley (Hordeum vulgare), highlighting the usability of this optimized sporulation protocol for the wider research community.Here, we optimized a multitude of parameters and report a sporulation method for Large-scale spore infection and virulence assays are essential for the screening of wheat and barley cultivars and combined with the genetic mapping of these populations allows pinpointing and exploiting sources of host genetic resistance. We anticipate that improvements in spore numbers and purity will further advance research to increase our understanding of the pathogenicity mechanisms of these important fungal pathogens.The online version contains supplementary material available at 10.1186/s13007-021-00751-4. Pyrenophora tritici-repentis (Ptr) which feeds and kills off the host tissue, thereby forming the characteristic necrotic and/or chlorotic spot-like lesions [Ptr has also been isolated from other cereal crops such as barley and other grasses [Ptr and its hosts as to reduce the damage caused by this economically important plant pathogen.Tan (syn. yellow) spot (TS) is an economically important disease of wheat and causes significant crop losses worldwide . The cau lesions . Yield l lesions , 3–5. Mo grasses , 7. ThisPtr secretes necrotrophic effectors (NE) during infection to kill the host tissue [Ptr, namely ToxA, ToxB and ToxC [Tsn1, Tsc2 and Tsc1 that interact with ToxA, ToxB and ToxC, respectively [Ptr [Ptr into race 1 to 8 is based on the Ptr NE profile and their disease scoring in these three differential wheat lines  [Ptr-wheat pathosystem and the establishment of a global race structure now allows for the screening of host genetic resistance and the development of more effective and sustainable control measures [t tissue . In accot tissue , 10. Thuand ToxC , 11. Wheand ToxC . ToxA inand ToxC , 15. Theectively . Throughely [Ptr . The clameasures .Ptr isolates across different races. For example, a high quality Ptr reference genome of the Australian race 1 isolate M4  was assembled and annotated using PacBio sequencing technology [Ptr isolates, including the race 5 DW5 isolate (ToxB) from the USA, were sequenced using Illumina paired-end reads [Ptr an ideal species for virulence studies.Besides the progress made in untangling the complex NE regulation , 20, valchnology . In addind reads . Along wnd reads , 19, thePtr were studied [Ptr conidia. However, this approach remained time-consuming and labour-intensive to harvest sufficient conidia numbers for applications such as infection assays [5 conidia in 20 ml of spore suspension) for infection studies. Despite the high inoculum number, the production of conidia using live plants from stubble-borne infection is time intensive and non-axenic, thus run the risk of containing co-infecting fungal pathogens of wheat [Inoculation experiments with asexual spores (or conidia) form a cornerstone in the research field of many fungal-plant pathosystems. This includes the need to carry out disease resistance screening , 22, com studied . Odvody  studied  reported studied  by inclu studied  formulatof wheat .Ptr that is simple, time-efficient and can consistently generate high spore inoculum numbers, free of mycelial/microbial contaminations. To efficiently harvest the conidia raised through this optimised approach, we devised a simple harvesting strategy by taking advantage of the hydrophobic property of fungal conidia. By adopting these modified approaches, we were able to devise a simple protocol that repeatedly generates three orders of magnitude more conidia per vegetative petri dish (3 × 106) compared to the Lamari and Bernier-based method [Ptr isolates. We also tested this improved method on taxonomically related fungal pathogens of barley that causes net form diseases of barley, Pyrenophora teres f. teres and f. maculata, and similarly observed significant improvements in conidia production and purity.In this study, we set out to develop an optimized sporulation protocol using in vitro culturing of d method . MoreovePtr spore numbers and avoid mycelial contamination would be desired. Currently, the routinely used method described by Lamari and Bernier [Ptr spores free of mycelial contamination, we tested a multitude of parameters at each of the different stages involved in sporulation. A focus on five main variables was established, covering the vegetative growth stage, conidiophore and conidia production as well as spore recovery and storage methodologies  as well as the number of transferred discs, the placement of the discs and even the type of media of the transfer plate , all of which resulted in different final spore numbers. The full set of tested values for each parameter is described in Additional file Ptr spore numbers. Conidia are hydrophobic due to surface proteins such as hydrophobins and adhesins which aid in their dispersal and host immune evasion as well as mediating host adherence [A major focus for developing the harvesting technique was on the increased quantity and purity of spores, which would subsequently lead to improved utility and reproducibility for downstream applications. We split the spore recovery stage further into six smaller categories to allow a step-by-step testing of multiple parameters within the pre-harvest preparation, the choice of flooding solution, the release, extraction, purification and concentrating method . Taken together, these numbers support our method as a robust and reliable sporulation protocol for Australian Ptr isolates.Next, we wanted to validate this protocol for other Australian on Table . One isoPtr community so next we tested the ToxB-producing DW5 race 5 strain from the USA, which also has excellent genomic resources available, making it the ideal testing candidate [Ptr spores was repeatedly exceeding one million compared to an average of 1500 spores that we were able to isolate using the Lamari and Bernier method [Ptr isolates since collectively, the M4 and DW5 strains produce all three known effector molecules  thus far characterized in Ptr.We aimed to develop a protocol for the broader andidate . Originar method . This moPtr. Therefore, mycelial contamination should be avoided so spore counts can be a robust measure for input biomass.Reliable and consistent quantification of input material is crucial to perform comparative wheat infection studies with With the traditionally used method , myceliaPtr, we then examined harvested conidia by determining their germination rate through the formation of germ tubes. Ptr M4 and DW5 isolates were used and germination lagged in the DW5 spores. Whilst 45% of the M4 spores germinated within 30 min, DW5 only reached this germination percentage after 75 min  and Pyrenophora teres f. maculata (Ptm) causing net form net blotch and spot form net blotch in barley, respectively. Compared to their traditional isolation methods, Ptt spore numbers were increased a 1000-fold whereas Ptm spores increased over 160 times . This further highlight we provided a useful tool to accelerate Pyrenophora research aiming to better understand and manage the impact of these globally devastating fungal pathogens.This study presents a sporulation method that permits high, pure and infectious spore isolations of important fungal necrotrophic phytopathogens of wheat and barley. This sporulation protocol details every optimized parameter and introduces new ways of harvesting conidia to avoid mycelial contamination. Moreover, this protocol can be completed within just seven days and this includes the vegetative growth stage of the pathogen. Spore inoculum levels were increased significantly (> 1000-fold) for both the M4 and DW5 Ptr pathogenicity and virulence or to screen for novel sources of resistance, large spore inoculums are typically required. We succeeded in developing a sporulation protocol that minimizes space occupancy and time and maximizes spore numbers absent from mycelial contamination. Currently, the most common and widely used Ptr sporulation method dates back to 1989 and usually only yields between 1000 to 3000 spores per vegetative growth plate [Ptr by testing and adapting previously reported sporulation methods and including new techniques. We focused on five main stages during sporulation and assayed a multitude of parameters within each of these pillars  were included in this study to assess the usability of this protocol across related fungal plant pathogens. All isolates produced conidiophores after N-UV exposure, which is visible as a blackened outer rim on the mycelial discs  a subsequent dark period incubation is needed for Ptr sporulation [Ptr conidia which is seen as white translucent specks at the end of the conidiophores  all isolates now produced on average a minimum of one million spores per plate, which is a welcome improvement to the traditional yields. profile . We compt of Ptr . Two othscs Fig. . Whereasscs Fig.  incubaterulation . Dark inres Fig. c, d. ForM4 Table . With thPtr spore solutions with the development of the typical necrotic/chlorotic lesions  in 2009 [Ptr race 5 DW5 isolate was collected from North Dakota (USA) in 1998 [Ptr isolates were collected from four different states, namely one isolate from South Australia (16 FRG 175), four isolates from Western Australia , one isolate from New South Wales (17 FRG 135) and one isolate from Victoria (15 FRG 239). These were collected over multiple years ranging from 2015 till 2017 and are all race 1 isolates (containing Tox A and Tox C effectors) (Table Pyrenophora teres f. teres (Ptt) strain collected from the Wongan Hills in Western Australia in 2009 whilst SG1 is a Pyrenophora teres f. maculata (Ptm) isolate from Badgingarra (WA) collected in 1996 [Isolates were inoculated from mycelial stock onto 90 mm polystyrene petri dishes  filled with V8-PDA and maintained under a white light on a 12 h light: dark cycle for 5 to 7 days at 22–24 °C. V8-PDA contains 15 g/l agar, 10 g/l Potato Dextrose Agar, 150 mL/l V8 Juice and 3 g/l CaCO in 2009 . The Ptr in 1998 . M4 and  in 1998 . The loc in 1996 .3). Vegetative growth was maintained under a white light on a 12 h light: dark cycle for 5 to 7 days at 22–24 °C until mycelia were nearing the edge of the petri dish (between 70 and 80 mm in diameter).Isolates were inoculated from mycelial stock, stored at − 80 °C as freeze dried plugs, and single plugs were placed onto 90 mm polystyrene petri dishes  filled with V8-PDA . A single vegetative plate yields approximately 48 plugs and the bevelled edge of the tip maximises mycelial wounding which in turn promotes conidiophore formation. Sixteen plugs were then transferred to a fresh V8-PDA plate and arranged in an evenly spaced ring around its edge approximately 5 mm apart per fresh plate, giving a ratio of 1 vegetative: 3 sporulation plates. These sporulation plates were immediately transferred to an incubator at 22–26 °C, and irradiated for 24 h under a continuous near-ultraviolet (N-UV) light source emitting between 290 and 400 nm wavelength . Lids were removed from the petri dishes to maximise oxygen flow and the N-UV irradiation will result in the development of conidiophores in the form of a thick black ring around the plugs outer face, which can be confirmed through stereoscopic analysis. Once conidiophores were visually detected, lids were replaced, though not sealed, and incubated in the dark at 15 °C for 18 h, which induces conidia formation.Initially, plates were checked for conidial development under a stereoscope. Once spore maturity was confirmed, excess agar was removed from the plate by using a 14 mm cork borer to cut secondary plugs around the sporulating plugs, being careful not to touch any spores during excision. Following excision of excess agar, the remaining agar plugs were flooded with 5 ml of 0.02% Tween 20 . To maximise conidial release, the petri dish was tilted to achieve complete submersion of a single plug and the surface and edges of the primary submerged plug brushed in a circular motion with a WestArt 663 Sable Flat No.4 paintbrush. Brushing continued until the air trapped by the hydrophobic conidia was released and then repeated on each subsequent plug in the same manner, ensuring submersion was maintained at each step. g for 5 min and left to sit at room temperature until all conidia are settled before removing the pellet with a 1 ml pipette with the tip aperture cut to a diameter 2 mm to ensure easier pipetting of conidial masses.Once all plugs were brushed, all remaining agar and mycelia was removed from the dish leaving only the spore solution, which was then poured into a 50 ml Falcon tube . Spore suspension was spun at 211Spore solutions were quantified by streaking out 3 × 10 µl of spore solution on a superfrost glass microscopy slide  and counting manually with a counter clicker under a Nikon eclipse E200 microscope (10X magnification). To assess spore viability, the germination rate of three diluted spore suspensions, each containing 35 spores, was followed over time until 100% germination rate was reached. Germination was counted under the Nikon eclipse E200 microscope (4X magnitude) where a positive count equals a spore with a protruding germination tube.Ptr bioassays, the second leaf of 14-day-old wheat seedlings (cultivar Axe–rated as TS sensitive) was attached, adaxial side up, to corrugated sheeting using microporous surgical tape . Leaves were inoculated with an evenly sprayed spore suspension at a concentration of 1 × 105 spores/ml in 0.02% (v/v) Tween 20 using the head from a 0.5 Trigger Sprayer . As a negative control, leaves were attached and sprayed with a 0.02% (v/v) Tween 20 solution as described above. Leaves were allowed to dry before being released from the micropore tape and the wheat seedlings incubated in a controlled growth chamber  under a 12 h light period at 90% relative humidity and 22 °C with regular watering intervals every 4 h. Lesions typically start to form from 3 days post inoculation.For Additional file 1: Figure S1. Collection sites of seven Australian Ptr isolates from the field. Each collection site is depicted as a grain pictogram with the different colours representing isolates from different states. The exception is Western Australia, where the three blue Ptr strains were isolated from wheat whilst the yellow site represents a Ptr strain isolated from barley. All other Ptr strains were isolated from wheat. This figure was created using Map Data © 2021 Google. Table S1. Growth conditions tested for conidia production in Ptr. Table S2. All harvesting parameters tested for purity and concentration in Ptr Optimal values are underlined.


COVID 19 is associated with a hypercoagulable state and frequent thromboembolic complications. For how long this acquired abnormality lasts potentially requiring preventive measures, such as anticoagulation remains to be delineated. We used viscoelastic rotational thrombelastometry (ROTEM) in a single center cohort of 13 critical ill patients and performed follow up examinations three months after discharge from ICU. We found clear signs of a hypercoagulable state due to severe hypofibrinolysis and a high rate of thromboembolic complications during the phase of acute illness. Three month follow up revealed normalization of the initial coagulation abnormality and no evidence of venous thrombosis in all thirteen patients. In our cohort the coagulation profile was completely normalized three months after COVID-19. Based on these findings, discontinuation of anticoagulation can be discussed in patients with complete venous reperfusion. It causes coronavirus disease 2019 (COVID-19) which often can take an asymptomatic course but can also result in substantial severe manifestations, such as acute respiratory failure, acute kidney failure, multi-organ dysfunction and death. It has led to a global pandemic with over 2.9 million attributable death toll to date6.While severe respiratory failure seems to be the most frequent cause of death, other complications like acute kidney injury, cardiac and neurologic involvement seem to occur more frequent than initially expected11. Laboratory markers revealed hyperinflammation linked to a hypercoagulable status with markedly elevated levels of fibrinogen, d-dimers and thrombocytosis. Numerous haemostatic tests performed in ICU patients with COVID-19 have shown, namely viscoelastic tests , Thrombin Generation  and Clot Waveform analysis  have demonstrated a hypercoagulable state16.From the beginning of the pandemic, a high incidence of thromboembolic complications was reported in patients with COVID-19 and autopsy findings confirmed a high rate of local thrombosis and embolic events in the pulmonary and systemic circulation associated with the disease17. In this context the term fibrinolytic shutdown has been proposed18.In our intensive care units we performed viscoelastic testing and noted severely impaired fibrinolysis as a characteristic of procoagulation in COVID-1923. The question for how long survivors of the disease should receive subsequent anticoagulation as secondary prophylaxis is unclear so far. Long term data regarding the question whether the hypercoagulable state persists after clinical cure of the disease are currently lacking23.These findings led to intensive discussions about the pros and cons of systemic anticoagulation, use of available drugs and intensity of systemic anticoagulationHere we present observational data of 13 critically ill patients with COVID-19 who presented at the post Intensive Care clinic of a tertiary care university hospital 3 months after discharge from ICU.17. All of these were invited for a follow-up visit. Thirteen patients came back to the follow-up visit after 3 months.Out of 41 Covid-19 patients admitted to our ICUs between March 25th and May 11th 2020, 29 patients survivedUpon admission on the ICU, all COVID patients were treated with therapeutic anticoagulation using intermediate doses of unfractionated heparin or argatroban with a aPTT goal of 50–55 s. Nevertheless, nine out of thirteen patients developed thromboembolic complications during their stay on the intensive care unit (ICU). These patients were then anticoagulated using higher doses of unfractionated heparin or argatroban with a targeted partial thromboplastin time of 60–80 s, respectively. Upon transfer to rehabilition units the continuation of this therapy regimen was recommended followed Rivaroxaban 20 mg per day until the first visit to the follow up clinic 12 weeks after discharge from the ICU.24. In each patient, intrinsically  and extrinsically  activated test assays were performed to analyze the clot dynamics in both coagulation pathways. Furthermore, FIBTEM and HEPTEM were performed. For FIBTEM thrombocytes are inactivated with Cytochalasin D to allow isolated evaluation of fibrinogen in clot firmness. For HEPTEM heparinase is added. The heparin effect was determined by comparing the clotting time of the INTEM with the clotting time of the HEPTEM.After admission to our ICUs, routine viscoelastic tests were performed with citrated blood by using a ROTEM sigma point-of-care device The following ROTEM variables were analyzed: clotting time defined as the time until initiation of clotting; clot formation time (seconds until a clot strength reaches 20 mm), reflecting the kinetics of clot formation; maximum clot firmness (MCF) defined as the maximum amplitude of clot firmness; maximum lysis (ML) in percent (%) defined as the difference between MCF and the lowest clot amplitude after MCF, reflecting fibrinolytic activity Fig. .Figure 1Additional routine laboratory tests carried out according to standardized protocols comprised haemoglobin concentration, white blood cell count, platelet count, prothrombin time (PT), International Normalized Ratio (INR), activated partial thromboplastin time (aPTT), d-dimers, fibrinogen, Interleukin 6, CRP and Ferritin  to screen for deep venous thrombosis, focusing on the jugular, subclavian, brachial, femoral and popliteal veins upon admission to our ICU and subsequently at least once a week.The same examinations were performed as part of the 3 months follow-up visit.All patients were invited for follow-up visits scheduled 3 months after discharge from the ICU. Follow-up visits took place at our post-ICU outpatient department and followed a standardized procedure including a medical consultation, ultrasound examination of the large vessels as described above and routine laboratory work up, including vioscoelastic tests.The study was approved by the ethics committees of Charité—Universitätsmedizin Berlin (EA4/115/20) and was in compliance with the Declaration of Helsinki. Consent to participate was not applicable due to the retrospective nature of the study. Informed consent was exempted by the ethics committee of the Charite Universitätsmedizin Berlin (EA4/115/20).Statistical evaluations were performed with IBM SPSS Statistics Version 26  and GraphPad Prism . Descriptive variables were given as median with limits of the interquartile range (IQR) for continuous variables or as absolute and relative frequencies for categorical variables.Continuous data were mostly right skewed. Therefore, Wilcoxon signed rank test was used to compare changes in continuous variables between ICU stay and 3 months follow-up evaluation. A two-sided significance level of 0.05 was applied without adjustment for multiple comparison. All p-values constitute exploratory data analyses and do not allow for confirmatory generalization of results.Out of 41 Covid-19 patients admitted to our ICUs between March 25th and May 11th 2020, thirteen patients came back to the follow-up visit after three months. Out of 13 patients, 9 were male with a median age of 60 [IQR 53–67] years and a median BMI of 29.6 [IQR 27.8–33.9].Their median SOFA score was 10.0 [IQR 5.3–11.8] points and their median APACHE II was 32.5 [IQR 25.3–34.0] points. Eleven patients required mechanical ventilation (85%), whereas extracorporeal membrane oxygenation was required in five (39%). Nine patients developed acute renal failure requiring continuous renal replacement therapy (69%). Median length of stay in the intensive care unit was 45 [IQR 30–65] days. , whereas prothrombin time and INR revealed no significant difference between measurement time points. CRP , Ferritin  and IL-6  were significantly elevated during the ICU stay.In terms of the measurements of coagulation values, patients during their initial ICU admission had significantly elevated levels of d-dimers  and fibrinogen  compared to their follow-up measurements  in INTEM and EXTEM increased significantly until 3 months follow-up  marking a substantial normalization of the fibrinolytic capacity which was markedly impaired at the initial assessments.During ICU stay, nine patients developed thromboembolic complications. In this regard, two patients developed pulmonary embolisms, while lower-extremity deep venous thrombosis was found in seven patients.At 3 month follow-up, there was no sonographic evidence of thrombosis in any of the patients indicating complete recanalization of the prior occluded veins  and histones on the endothelium45.Tang et al. demonstrated decreased mortality in COVID-19 patients with coagulopathy who were treated with either unfractionated heparin or low molecular weight heparin (LMWH) for 7 days or longer compared to those who did not receive heparin19.The American College of chest physicians (ACCP) and the American Society of hematology (ASH) recommended low molecular weight heparin (LMWH) in prophylactic doses in critical ill COVID-19 patients and in therapeutic dose if venous thromboembolism (VTE) occurred46.Recent guidelines of the International society on thrombosis and hemostasis (ISTH) recommend either prophylactic or intermediate dosing of LMWH or UFH47.Given the high incidence of thromboembolic events many centers switched to intermediate dosing of anticoagulation instead of standard prophylaxisAs a consequence, our patients received unfractionated heparin with a target PTT of 50–55  seconds as long as there were no thromboembolic complications. Patients with thromboembolic events received therapeutic doses of unfractionated heparin with a target PTT of 60–80 s. We decided against routine measurements of AT-III-levels in all patients, instead patients who received more than 1800 U of unfractionated heparin per hour and still did not reach their taget PTT were classified as heparin resistant and switched to Argatroban. The reason for this being the fear of increasing the risk of enhancing bleeding episodes by substitution of AT-III while at the same time further uptitrating the dose of unfractionated heparin, given the difficult situation of frequent thromboembolic complications on the one hand and reported enhanced risk of bleeding episodes on the other. We decided to use Argatroban and not Bivalirudin due to the greater experience in our department with the use of Argatroban. We did not encounter spontaneous prolonging of the aPTT and did not routinely check for lupus anticoagulans. Since the turnaround time for the anti-Xa-activity is about 12 h at our institution, we decided against its use for guiding our anticoagulation management.50.In contrast to individual parameters, viscoelastic methods like thrombelastography and ROTEM permit functional evaluations of whole blood aggregometry. Thus it allows evaluation of the different and complex coagulation phases including the initiation, formation and stabilization of a clot, and finally, clot lysis. Still endothelial function and the influence of soluble tissue factor have to be taken into account as they will not be represented in the results of the test. Not only bleeding diathesis as the classical indication for viscoelastic testing but also hypercoagulable conditions due to different diseases were examined in the past using ROTEM and states of hyper- and hypofibrinolysis could be reliably detected and characterized by viscoelastic testsOur cohort presented with a significantly increased clot firmness on the one hand and severely impaired fibrinolytic activity represented by a maximum lysis of < 3% during their ICU-stay on the other.14. It has also been proposed that decreased activity of urokinase-type plasminogen activator and increased release of plasminogen activator inhibitor-1 might be the mediating mechanism of hypofibrinolysis, but data to support this further are scarce51.The clot lysis parameter ML provides information on the fibrinolytic capacity and was successfully used in several studies to asses hyper-, or hypofibrinolysis. Lower values of ML provide evidence of existing hypofibrinolysis, while values above 15% are suggestive for hyperfibrinolysis. Other groups already demonstrated that global haemostatic tests in critical ill patients with COVID-19, tests revealed a hypercoagulable state. That holds true for viscoelastic tests like TEG and ROTEM as well as Thrombin Generation  and Clot Generation Waveform analysis . Nouigier et al. reported in their recent study that critical ill patient with COVID pneumonia have an impaired fibrinolytic capacity which was associated with increased levels of PAI-1 and TAFIOn their 12-week follow up visit, clot firmness and fibrinolytic activity had normalized in all patients. The significant increase of ML in the follow-up assessment indicates an appropriate regeneration or reversibility of physiologic fibrinolytic capacity.To the best of our knowledge, this is a first study reporting follow-up data on the reversibility of thrombelastometry abnormalities after COVID-19. We found normalized fibrinolytic activity and normalized clot firmness. In contrast, von Meijenfeldt et al. found sustained prothrombotic changes in COVID-19 patients 4 months after hospital discharge. 53Hemostasis exams showed enhanced thrombin-generating capacity and decreased plasma fibrinolytic potential. Further studies will be necessary to clarify this contradictory data situation.19. ISTH states, that post discharge prophylaxis for 2–6 weeks should be considered46. For patients after VTE, current guidelines recommend therapeutic anticoagulation for at least 3 months46.ACCP recommends to evaluate patients for extended prophylaxis after their hospital stay depending on their risk of bleeding52. Around 60% of all VTE in medical patients occur in the post‐hospital discharge period with a more than 5 times increased risk in fatal pulmonary embolism53. Newer studies reported favorable risk–benefit ratios for extendend prophylaxis in medical patients54.Especially patients with high d-dimer values were found to be at high risk of post-discharge VTE independent of COVID-19In conclusion our patients who suffered from thromboembolic events during their course of COVID-19 received therapeutic anticoagulation during their ICU stay and continuation was recommended for the first 3 months thereafter. Taking into account the grade of immobilization and the high levels of d-dimers, increased clot firmness and severely impaired fibrinolysis on viscoelastic testing, we recommended to evaluate therapeutic anticoagulation in the patients without thromboembolic complications at least until reassesement during their first visit on the post-ICU-clinic. On the other hand, proper patient selection to identify patients at higher risk for bleeding while at the same time weighing it against the risk of thrombosis is crucial.On their 3 months follow-up visit all patients presented with normalized values of d-dimer, fibrinogen and viscoelastic testing. Inflammatory markers were also normalized. Since there were no signs of a persistent hypercoagulable state left and none of the patients suffered from a thromboembolic event after discharge the discontinuation of anticoagulation can be discussed and future clinical trials are needed.Our study has several limitations. We report the data of a relatively small single center cohort of critically ill patients which may limit generalizability. Due to its retrospective nature it can only be hypothesis generating. Our presumptions have to be verified in a clinical trial focusing not only on coagulation profiles but also on clinical data such as rate of thromboembolic events and ideally survival.Furthermore, in the meantime between ICU discharge and follow-up, no control follow-ups were routinely performed or analyzed.Moreover, we only performed anamnesis, clinical examination and screening ultrasound examinations as follow-up exams.. There were no clinical or anamnestic hints for pulmonary embolism but routine tests regarding asymptmomatic events were performed so no statements regarding asymptomatic pulmonary embolism can be made.In summary, we found substantially limited fibrinolysis in acutely ill COVID-19 patients with normalization after three months.In our cohort of critically ill COVID-19 patients, the coagulation profile and inflammatory markers were completely normalized three months after discharge from the ICU. It thus appears reasonable that anticoagulation can be discontinued beyond this timepoint in patients with complete venous reperfusion.


B-cell receptor (BCR) signaling is crucial for the pathophysiology of most mature B-cell lymphomas/leukemias and has emerged as a therapeutic target whose effectiveness remains limited by the occurrence of mutations. Therefore, deciphering the cellular program activated downstream this pathway has become of paramount importance for the development of innovative therapies. Using an original ex vivo model of BCR-induced proliferation of chronic lymphocytic leukemia cells, we generated 108 temporal transcriptional and proteomic profiles from 1 h up to 4 days after BCR activation. This dataset revealed a structured temporal response composed of 13,065 transcripts and 4027 proteins, comprising a leukemic proliferative signature consisting of 430 genes and 374 proteins. Mathematical modeling of this complex cellular response further highlighted a transcriptional network driven by 14 early genes linked to proteins involved in cell proliferation. This group includes expected genes  and genes involved in NF-kB signaling modulation  and immune evasion  that have not yet been associated with leukemic cells proliferation. Our study unveils the BCR-activated proliferative genetic program in primary leukemic cells. This approach combining temporal measurements with modeling allows identifying new putative targets for innovative therapy of lymphoid malignancies and also cancers dependent on ligand–receptor interactions. In B-cell lymphomas and leukemias such as marginal zone lymphoma and chronic lymphocytic leukemia (CLL), chronic antigenic activation of the B-cell antigen receptor (BCR) sustains aberrant lymphocyte behavior and uncontrolled monoclonal proliferation . In physEx vivo models of BCR stimulation in patients’ primary CLL cells , 8–10 haIn the present study, we used this improved ex vivo culture model to generate a unique set of 108 combined transcriptional and proteomic profiles over time after activation of human primary CLL cells. As existing analysis methods of such high dimensional datasets are limited in terms of precision to select the relevant actors of the genetic program, we have developed a mathematical approach, validated on synthetic datasets and supported by extensive simulations, allowing the selection of critical actors of the CLL’s cell response . FurtherB cells from peripheral blood were obtained from six untreated CLL patients whose biological characterization, performed at the University Hospitals of Strasbourg, included flow cytometry analysis, cytogenetic with FISH, IGHV status, and TP53 mutational profile. All selected patients had a Matutes score of 5/5, unmutated IGHV, and wild-type TP53 Table . This st6 cells were resuspended in 1 mL TRIzol . Total RNA was purified using the RNeasy Mini kit . After ribosomal RNA depletion, the sequencing library was prepared with the Ion Total RNA-seq kit v2 (Thermo Scientific) and the sequencing was performed on an Ion Proton sequencer with the Ion PI Hi-Q Sequencing 200 Kit (Thermo Scientific). Reads was estimated with the package HTSeq [Before BCR engagement (T0) and at eight time points after activation , 4.10ge HTSeq  and the ge HTSeq  was used6 cells were resuspended in lysis buffer. Proteins were acetone precipitated and 10 µg of each sample were concentrated in a stacking gel band, in-gel reduced, alkylated, and trypsin digested. NanoLC-MS/MS analyses of extracted trypsic peptides were performed on a nanoAcquity UPLC device (Waters Corporation) coupled to a Q-Exactive Plus mass spectrometer (Thermo Scientific) operated in data dependent acquisition mode. Label-free extracted ion chromatogram-based quantification was performed using MaxQuant software (version 1.5.5.1) [Before BCR stimulation (T0) and at eight time points after stimulation , 8.101.5.5.1) . The proQuality-based filtering of low expressed genes was performed with the HTSFilter package . The selT = 8 time points and for a number of P = 3 individuals (3 proliferative and 3 nonproliferative samples); we denote by After selection according to their differential expression and temporal profile, genes and proteins were divided into temporal clusters for network reverse engineering. We had to model In this model, https://fbertran.github.io/Patterns/) [The code written for selection of actors and reverse engineering the temporal cellular program in this study is available as an R-package (tterns/) .Experimental procedure is summarized in Fig. To investigate the proliferative response of primary human CLL cells after ex vivo BCR engagement, BCR stimulation was performed on six untreated CLL samples of the most aggressive subgroup (IGHV unmutated), including three samples that proliferate and three samples that do not proliferate ex vivo within our culture conditions, the latter being used as controls Table . TranscrX axis and the proliferative status of the samples on the Y axis. The dots corresponding to the proliferative and nonproliferative samples were separated at T0 along the Y axis and they all followed a structured evolution from T1 to T8 along the X axis after BCR engagement. This analysis emphasized the structured nature of the transcriptional BCR response. In addition, hierarchical clustering analysis strengthened this structured nature since it identified four clusters, each made of consecutive time point of measurement . However, a tendency for samples’ segregation with respect of their proliferative response was observed at later time points (T7 and T8) Fig. . This waHaving evidenced the structured nature of the global CLL cell response to BCR activation, we next characterized the proliferative signature within this response, defined as the genes and proteins DE in the proliferative samples compared to the nonproliferative samples after BCR-mediated CLL cells activation. To determine this signature, we first established the list of genes and proteins significantly (FDR: <1%) up- or downregulated in the stimulated (T1–T8) versus unstimulated (T0) samples, defining a temporal signature Fig. . In the As expected, we observed a delay between the transcriptional and the translational response after cell stimulation. Indeed, as many as 1133 genes (511 upregulated and 622 downregulated) were already DE in proliferative samples in the 3 h after BCR stimulation Fig. , whereasWe next analyzed the function of the 1107 up- or downregulated proteins of the temporal signature in the proliferative CLL cells. Biological process annotations (GO BP terms) were collected in order to calculate the number of proteins involved in each process during the timecourse after cell activation. Albeit no significant proteomic enrichment was noticed at early time point, an increase in the number of upregulated proteins related to signaling, transcriptional activity, and cell activation was observed between 7 and 24 h (T4–T6), and a second upsurge of upregulated proteins related to signaling, metabolism, transcriptional processes, cell cycle regulation, DNA replication, nuclear division, and proliferation occurred from days 2 to 4 (T7 and T8) Fig. . The mosLooking at GO BP for downregulated proteins revealed a transiently decreased number of proteins related to signaling, metabolism, and differentiation within 1 h (T1) after cell activation, potentially reflecting a catabolism phenomenon Fig. . The numAltogether, this multiomic approach highlights the structured nature of the temporal response to BCR stimulation in primary CLL cells, characterized by an early transcriptional component progressively relayed by a proteomic component including elements related to the onset of cell proliferation.F-score, were validated with simulated data and compared to the performance of other algorithms  in the network Fig. . AnalyziThe first layer corresponds to the 173 seeding proteins associated with cell cycle regulation or proliferation processes. These proteins segregate within two groups according to their chronological involvement during the temporal response Fig. . The firThe second layer corresponds to the 71 genes present in the subnetwork that encode proteins of the first layer Fig. . RemarkaThe third layer of this subnetwork comprises 144 elements made of 50 genes and 94 proteins. At early time points (T1 and T2), only 19 elements are present of which 14 genes exhibit a strong upregulation corresponding to the very early transcriptional response to CLL cell activation. These genes encode major transcription factors (TFs) involved in the G0/G1 transition , in the regulation of B-cell proliferation, and differentiation after BCR activation  which are also involved in lymphomagenesis [Remarkably, the mathematical modeling of the temporal multiomic data allowed to trace back the sequential organization of the proliferative CLL response from the protein effectors of cell proliferation at day 4 to the early molecular events induced after BCR activation.We next investigated if this proliferative subnetwork composed of TFs and hub proteins is specific to the CLL proliferative program. For this purpose, we inferred with the same approach the formalized model with the temporal datasets of nonproliferative samples. This led to the identification of a regulatory network of 1399 genes and 750 proteins representing 1933 unique symbols, which were connected by 17,332 oriented links Table . FunctioThe search for neighbors of these 193 proteins revealed 131 elements with a nested subnetwork comprising 114 actors (37 genes and 77 proteins) Fig. . LookingThe characterization of the cellular program sustaining CLL cells proliferation after BCR engagement is a major step to understand mature B-cell leukemogenesis with the ultimate goal of developing innovative therapies targeting the nuclear response to BCR activation instead of the cytoplasmic pathways that can be bypassed in resistant cancer cells. However, studying the proliferative program in primary CLL cells is challenging because of the difficulty to experimentally recapitulate cell proliferation ex vivo. Here, we used the recently developed culture model based on BCR engagement to induce CLL cell proliferation  which alOur large multiomic study highlights in human primary cancer cells the coordination between the dynamic gene and protein responses after exogenous cell stimulation. So far, only few studies have addressed this relationship showing a relatively weak gene-to-protein correlation of 30–60% in yeast –31, muriRemarkably, the proliferative subnetwork identified by modeling the response of aggressive CLL lymphocytes to BCR activation with costimulating agents comprised several genes encoding important TFs downstream the BCR signaling pathway, including genes previously identified in common in the lymphocytic response to BCR alone in healthy donors and patients with indolent or aggressive CLL  Table S. This inAmong the TFs identified here in the response of aggressive CLL lymphocytes, EGR1 and EGR2 are zinc-finger TFs downstream of the Ras/Raf/MAP kinase pathway that is constitutively activated in various cancers and blood malignancies . EGR1 inFurthermore, this subnetwork suggests a prominent activation of immune-evasion mechanisms of CLL cells after BCR and associated factors mediated cell proliferation activation. The genes KMO and IL4I1, respectively, involved in tryptophan catabolism  and germNoteworthy, the nonproliferative cells response did not exhibit most of the above genes, validating the composition of the core subnetwork of the BCR-mediated cell proliferation. However, comparison of the 388 actors (genes or proteins) of the proliferative subnetwork Fig.  and the In conclusion, using a large dataset of temporal transcriptional and proteomic measurements coupled with mathematical modeling, this study unveils the genetic program downstream the signaling cascade activated by the BCR engagement and triggering primary CLL cell proliferation ex vivo. Noteworthy, this program organizes around a limited number of genes and proteins whose sequential commitment drives the cellular response leading to proliferation days after cell activation. These hubs represent potential targets for the development of novel therapeutic strategies for the treatment of aggressive CLL. Beyond CLL, such an approach could be explored in other mature B and T antigen-driven malignancies and could also be extended to other cancer types dependent on ligand–receptor interactions, as for instance the hormone-dependent cancers.Supplemental MethodsSupplemental Figures_RevisedTable S1Table S2_Revised


Despite low mortality, cardiac surgery patients may experience serious life-threatening post-operative complications, often due to extracorporeal circulation and reperfusion. Miniaturised cardiopulmonary bypass  has been developed aiming to reduce the risk of post-operative complications arising with conventional extracorporeal circulation.The COMICS trial is a multi-centre, international, two-group parallel randomised controlled trial testing whether type II, III or IV minimally invasive extracorporeal circulation is effective and cost-effective compared to conventional extracorporeal circulation in patients undergoing elective or urgent coronary artery bypass grafting, aortic valve replacement or coronary artery bypass grafting + aortic valve replacement. Randomisation (1:1 ratio) is concealed and stratified by centre and surgical procedure. The primary outcome is a composite of 12 serious complications, objectively defined or adjudicated, 30 days after surgery. Secondary outcomes (at 30 days) include other serious adverse events (primary safety outcome), use of blood products, length of intensive care and hospital stay and generic health status .Two centres started recruiting on 08 May 2018; 10 are currently recruiting and 603 patients have been randomised (11 May 2020). The recruitment rate from 01 April 2019 to 31 March 2020 was 40-50 patients/month. About 80% have had coronary artery bypass grafting only. Adherence to allocation is good.The trial is feasible but criteria for progressing to a full trial were not met on time. The Trial Steering and Data Monitoring Committees have recommended that the trial should currently continue. Morbidity occurs because surgery itself carries a risk of iatrogenic harm, primarily as a result of ischaemia reperfusion injury (IRI)4 Type IV hybrid modular MiECC systems (integrating a hard-shell venous reservoir as a stand-by component for immediate conversion to an open system) overcome safety concerns and unexpected intraoperative perfusion scenarios; thus, they are compatible with every cardiac surgical procedure.5Cardiac surgery with conventional extracorporeal circulation (CECC) provokes a vigorous SIR due to activation of stress pathways associated with post-operative end-organ complications . SIR is triggered by operative surgical trauma and IRI but is further exacerbated by the interaction of air, blood and synthetic components in the CECC apparatus. Minimally invasive extracorporeal circulation (MiECC) systems integrate contemporary innovations in CPB technology into a strategy which aims to reduce SIR and improve end-organ protection during cardiac surgery. They comprise a closed circuit with biologically inert blood contact surfaces and reduced priming volume; a centrifugal pump; a membrane oxygenator; a heat exchanger; a venous bubble trap or venous air removing device; a cardioplegia system and a shed-blood management device.69 ) have reported substantial benefits of MIECC for 30-day mortality, stroke, myocardial infarction (MI), post-operative atrial fibrillation and renal dysfunction. No harms were reported. The diversity of MiECC systems, patients and outcomes, and the poor methodological quality of many of the randomised clinical trials (RCTs) included in these reviews, undermines the strength of this evidence and most hospitals continue to use CECC.9 Nevertheless, the potential benefit of MiECC highlights the urgent need to evaluate this technology in a large, high-quality RCT. The proposed trial will evaluate MiECC systems that conform to an established typology,5 recruit patients having the most common cardiac surgery operations, report important clinical outcomes and includes features to minimise bias.Four meta-analysesThis COMICS trial is an international, multi-centre, randomised, controlled parallel group trial to investigate the effects of using MiECC in all patients having elective or urgent coronary artery bypass grafting (CABG), aortic valve replacement (AVR) or CABG + AVR using extracorporeal circulation without circulatory arrest . The triThe trial has three specific objectives:To estimate the difference between groups in the proportion of participants experiencing the primary outcome (a composite of serious complications) up to 30 days after randomisation.To compare secondary outcomes between groups: serious adverse events  not included in the primary outcome, red blood cells (RBC) and other blood products transfused; duration of cardiac ICU and hospital stay following the index admission; resource use, generic health status.To estimate the cost-effectiveness of MiECC compared to CECC.The trial is recruiting patients undergoing CABG, AVR or CABG + AVR. Cardiac surgery centres in Germany, Greece, Italy, Saudi Arabia, Switzerland, Turkey and the United Kingdom are participating. Additional centres in the United Kingdom, Germany and Canada are in set-up.Patients are eligible if all the following apply:Age ⩾18 and <85 years.Undergoing any elective or urgent CABG, AVR or CABG + AVR surgery, using extracorporeal circulation without circulatory arrest.Patients are ineligible if any of the following apply:Requirement for emergency or salvage operation.Requirement for major aortic surgery (e.g. aortic root replacement).3 Contraindication or objection (e.g. Jehovah’s Witnesses) to transfusion of blood products.Congenital or acquired platelet, RBC, or clotting disorders (patients with iron deficient anaemia only are not excluded).Inability to give informed consent for the trial (e.g. learning or language difficulties).Potential trial participants are identified before their surgery from clinic lists and inpatient referrals. All potential participants receive an invitation letter and a patient information leaflet (PIL) describing the trial and are given time to discuss their participation with others outside the research team (e.g. relatives or friends) if they wish. Before their operation, patients are seen by a member of the local research team answers any questions, confirms eligibility and takes written informed consent if the patient is willing.4 Centres may use any MiECC circuit which uses CE-marked components (or the country-specific required standards outside the European Community) and which have features consistent with Type II, III or IV criteria.MiECC systems have evolved in a modular fashion, to address safety, volume and blood management issues. Systems have been classified according to their features .CECC should comprise (required components) standard oxygenator, roller pump, hard-cell reservoir, arterial filter, shed-blood suctions, any of a range of venting options, uncoated tubing and a cell-saver device. The following optional/alternative components can be integrated (and recorded accordingly): coated oxygenator, coated tubing and centrifugal pump. The following components are prohibited: soft-cell reservoir and vacuum-assisted venous drainage (these are advanced components which make CECC resemble a custom-made MiECC circuit).Other aspects of the operations may vary by operation and centre but must be used consistently in the MiECC and CECC groups. For example, surgeons may use different cardioplegia solutions at different temperatures in different centres or for different operations. However, a surgeon carrying out one type of operation in one centre, for example, CABG in Centre A, must use the same cardioplegia solution for both MiECC and CECC. We are collecting operative details to characterise and report these variations. We believe such diversity in practice in the trial will create greater confidence in the applicability of the findings to a potential user’s clinical setting.Randomisation is performed using a secure Internet-based randomisation system,  and is stratified by centre and surgical procedure . Participants are allocated in a 1:1 ratio to either MiECC or CECC. Randomisation takes place as close to surgery as possible and is performed by an authorised member of the local research team. Information to identify a participant and to confirm eligibility must be entered before a number is assigned .10 Participants are blinded to the allocation. Documentary evidence is being sought wherever possible for events that qualify for the primary outcome; other events will be adjudicated. The 30-day primary outcome minimises loss to follow-up. Reporting bias will be minimised by a statistical analysis plan finalised before the database is locked.The trial includes features designed to minimise bias.The primary outcome is a composite of post-operative SAEs occurring up to 30 days after randomisation following the index admission. All SAEs that qualify for the primary outcome are objectively defined or will be adjudicated. The following events qualify:(a) All-cause mortality up to 30 days after surgery(b) Myocardial infarction (MI)(c) Permanent stroke(d) Gut infarction11(e) AKI  Network criteria for stage 3 AKI(f) Reintubation(g) Tracheostomy(h) Mechanical ventilation for >48 hours, including multiple episodes when separated by more than 12 hours(i) Reoperation(j) Percutaneous intervention(k) Sternal wound infection with dehiscence(l) Septicaemia confirmed by microbiologyEvents will be documented as follows: suspected MI by serum troponin concentrations and electrocardiograph recording (ECG); stroke from brain imaging (CT or MRI) report and record of new onset focal or generalised neurological deficit; gut infraction by laparotomy or post-mortem; other events from the medical record. Equivocal events will be adjudicated.Secondary outcomes are as follows:(a) All-cause mortality up to 30 days after surgery(b) Other SAEs up to 30 days after surgery (primary safety outcome)(c) Units of RBC transfused up to 30 days after surgery(d) Other blood products transfused up to 30 days after surgery(e) Time to discharge from cardiac ICU during the index admission(f) Time to discharge from hospital following the index admission12 for up to 5 days (only collected at some centres).(g) Delirium in ICU, assessed with the Intensive Care Delirium Screening Checklist (ICDSC)13 at 30 and 90 days after randomisation(h) Generic health status using the EuroQol 5-level health status questionnaire (EQ-5D-5L)(i) Health resources and associated costs up to 90 days after randomisation (UK centres only)Participants are followed up twice, at 30 days and 90 days after surgery. Questions elicit information about SAEs (including readmissions) experienced since discharge up to 30 days after the date of surgery. Generic health status is assessed around both 30 and 90 days. The schedule of data collection is outlined in 14 Pooled estimates from previous meta-analyses of the effects of MiECC versus CECC have reported benefits of MiECC for a range of clinical outcomes: odds ratios for 30-day mortality, 0.586 0.407 0.46;9 for stroke, 0.256 0.407 0.40;9 for MI, 0.33,7 0.40;9 for post-operative atrial fibrillation, 0.62;9 renal dysfunction 0.47.9 A smaller but still clinically important target relative difference, that is, risk ratio of 0.75, was chosen for the trial. In order to detect a risk ratio of ⩽0.75 with 90% power and 5% significance (two-tailed), 2,504 to 3,258 participants are required, depending on the numbers recruited to different surgical strata. We propose to recruit 3,500 participants to allow for uncertainty in the assumptions underpinning this calculation.The trial is powered for the primary composite outcome. The transfusion indication threshold reduction 2 trial (TITRe2) trial, carried out in 17 UK centres, showed a rate of 15-18% in the post-operative SAEs qualifying for the primary outcome in participants receiving CECC, depending on the type of surgery received .SAEs are considered in three categories: SAEs that qualify for the primary outcome, other SAEs that are known complications of cardiac surgery, and SAEs that are not known complications of cardiac surgery. The latter will be subject to expedited reporting to the Lead Coordinating Centre. Expedited reporting will also include all fatal events.UK sites have ethics approval through the UK Integrated Research Application System (222991). Non-UK sites or countries are responsible for following their own local governance processes and obtaining the necessary approvals. The University of Bristol is acting as the sponsor for UK sites and as the central coordinating centre for non-UK sites.15 and signed off before any analysis takes place.Statistical analysis is the responsibility of the Bristol Trials Centre (BTC). A final statistical analysis plan will be written, consistent with the CONSORT guidelines, including the extension for non-pharmacologic treatment interventions,Analyses of all outcomes apart from the primary safety outcome will be conducted according to the intention-to-treat (ITT) principle, with participants analysed according to the groups to which they were randomised. A per-protocol (PP) analysis, only including participants who received the intervention to which they were allocated, will be considered for the primary outcome if there are a considerable number of protocol deviators. The safety population will consist of all participants, where participants will be included according to the intervention they received.The primary outcome will be assessed using a generalised linear model (GLM) to compare trial arms in terms of the risk ratio of experiencing a qualifying SAE up to 30 days after surgery, adjusting for centre and operative procedure.Binary secondary outcomes  will be assessed separately, using GLMs as for the primary outcome. The total number of units of blood products transfused will also be analysed using linear regression, adjusting for centre and operative procedure, for all blood products.Time to death, time to discharge from CICU and time to discharge from hospital will be assessed using Cox proportional hazards models to compare the trial arms, adjusting for centre and operative procedure, for both analyses. Median survival with 95% confidence intervals will be estimated by trial arm and overall for both analyses.Generic health status (EQ-5D-5L) will be analysed using a linear mixed effects model. Mean QoL scores and 95% CIs will be summarised at baseline, 30 days and 90 days.Occurrence and frequency of all safety events  will be summarised by trial arm and overall.Subgroup analyses will be carried out for the primary outcome to compare the trial arms across each level of the following characteristics:Age (dichotomised by the median)Sex Operation type EuroSCORE (dichotomised by the median)Preoperative renal dysfunction (dichotomised by the median)Preoperative Hb (dichotomised by the median)All subgroup analyses will be interpreted with caution and treated as hypothesis-generating.16The cost-effectiveness of MiECC compared to CECC will be estimated for COMICS participants recruited in England (approximately 600 participants), using quality-adjusted life years as the outcome measure estimated using the EQ-5D-5L, which can be mapped on to ‘valuations’.13 We will calculate average costs and outcomes associated with MiECC and CECC and produce an incremental cost per quality adjusted life year (QALY) gained with MiECC compared to CECC. If there are differences in mortality between trial arms, then costs and outcomes will be extrapolated to a longer time horizon. Scenario analyses will use EQ-5D-5L data from all participants and cost data from English participants.Costs of resource use for English participants will be estimated using linked Hospital Episode Statistics (HES) data and information from a previous trial.Two centres started to recruit on 08 May 2018. Ten centres are currently recruiting; another centre recruited for 14 months but stopped participating for reasons unrelated to the trial. About 40-50 patients were randomised per month from 01 April 2019 to 31 March 2020 with over 600 now randomised. About 80% of participants have had CABG only. Primary outcomes are available in the database for 85%, recognising that these may still have to be entered for recent recruits. Adherence to randomised allocation is good (99%).There have been significant challenges in setting up the trial and achieving the rate of recruitment required to complete the trial in a feasible duration (scheduled to recruit for 3 years from September 2020). A joint meeting of the Trial Steering and Data Monitoring Committees reviewed progress against criteria for continuing to the main trial in December 2019, 18 months after starting the pilot: ⩾16 centres recruiting; ⩾750 participants randomised; >90% adherence to the allocated intervention. These criteria were not met, primarily due to fewer than expected centres joining the trial especially during 2018. Nevertheless, the Committee chairs recommended continuing for a further 6-9 months, aiming to increase the recruitment rate and obtaining funding for the main trial. Additional centres are currently being set up and an application for funding has been submitted. New centres are welcome to join the trial (please email the contact address provided for further details).Negotiating contracts with each participating site has been the most serious challenge. The contract describes indemnity arrangements, key responsibilities of different parties, intellectual property and data ownership, data sharing and publication policy, even if no funding is offered. The main obstacles have been indemnity (the coordinating centre is not providing any), intellectual property ownership (owned by the coordinating centre) and the legal system under which any disputes would be considered (both governing law and jurisdiction), which have prevented some centres from taking part.Some centres have been unable to take part because there is no funding yet to reimburse their research costs; one potential centre considered this unethical. Award of funding would address this challenge and potentially increase recruitment in centres already recruiting.17The capital cost of adopting MiECC has been a barrier for centres wanting to use it. Adopting MiECC also requires entire surgical teams  to agree. MiECC also has higher consumable costs, although these may potentially be offset by clinical benefits (see above). European guidelines recommend that MiECC should be considered instead of CECC to increase the biocompatibility of extracorporeal circulation technology used in cardiac surgery .The COMICS trial is already the largest randomised trial of MiECC compared to CECC in patients having most cardiac surgery operations requiring extracorporeal circulation without circulatory arrest. The COMICS trial will have substantial health and socioeconomic impacts. It will quantify the effects of MiECC versus CECC on a range of important clinical outcomes , health resource use , health-related quality of life (EQ-5D-5L) and QALYs.