In 2015, a quality improvement project called “Projeto Parto Adequado-PPA” was implemented in Brazilian private hospitals to reduce unnecessary high rates of cesarean sections. This study aimed to analyze the decision-making process of managers and care leaders to adhere to the PPA.The Healthy Birth study is evaluative research that used mixed methods to evaluate the implementation and effects of the Adequate Childbirth Program in 12 hospitals that participated in the program. Eight out of 12 hospitals were selected for a qualitative approach. We interviewed ten managers and 24 care leaders from July to October 2017. The interviews were transcripted, and data was systematized using the MaxQda software, with Thematic Content Analysis, to identify the facilitators and barriers for adherence to the Adequate Childbirth Program. We used the conceptions of the Diffusion of Innovation as an analytical reference.The main reasons to adhere to the Adequate Childbirth Program were the absence of other quality improvements programs in Brazilian private services using multifaceted interventions, social and market status for participating; commitment to quality of care; and the possibility of structural reforms related to the Adequate Childbirth Program implementation. In addition, inviting hospital influencers to learn about the objectives and intentions of the project before joining was considered an important strategy to motivate hospitals.Social, cultural, and economic constructs motivated adherence. The invitation strategy used by the Adequate Childbirth Program coordination, through socially respected members in Brazil, such as doctors, was highly valued by the leaders of the hospital team and encouraged adherence to the Program. In 2015, a quality improvement program, called the "Adequate Childbirth Program" (PPA), was recommended by the National Supplementary Health Agency so that Brazilian private hospitals could, through a change in the care model, reduce unnecessary cesarean sections and stimulate the natural and safe process in childbirth. The process of adhering to the PPA was studied in this article. Ten managers and 24 assistant leaders were interviewed in 08 private hospitals in Brazil. The main reasons for joining the PPA were the absence of other quality improvement programs; for being a program considered necessary in the country's midwifery, which could increase the prestige of hospitals in the view of women and families; for proposing an improvement in the quality of care; and for stimulating of structural and ambiance reforms. Social, cultural, and economic values motivated hospital adherence. The invitation strategy used by the PPA coordination, through socially respected members in Brazil, such as medical professionals, was highly valued by the hospital's leaders and encouraged adherence to the Program. In Brazil, the logic of health system organization, typical of a capitalist state, conjugates different practices and care models. The Brazilian Unified Health System (SUS) combines public and private subsectors . The chaUnsatisfactory health outcomes, such as high cesarean rates in the Brazilian private sector, require sophisticated solutions with great inventive potential. In the private sector, cesarean section accounts for 83 percent of births , 5, otheIn 2006, an extensive movement of women, called “Parto do Princípio”, began the struggle for rights related to childbirth and birth in Brazil. Since then, it has been present in the fight against obstetric violence and is presented essentially in the scenario of dialogue in the health system to adopt measures that quality care and reduce the high rates of cesarean sections .The “Parto do Princípio” movement presented a dossier to the Federal Public Ministry proposing changes in the Brazilian obstetric private sector in 2006 . In 2010In this sense, such reality induced in 2015 the creation of the Adequate Childbirth Program (Projeto Parto Adequado—PPA), which is an intervention proposed by the National Agency for Supplementary Health (ANS), to improve delivery and birth care in Brazilian private hospitals. In which it proposed to identify innovative and viable models of care, based on successful experiences and scientific evidence, in an attempt to reduce the potential increase of unnecessary risks of cesarean sections, insecurity, and experiences that do not reveal the empowerment and protagonism of women in the scenario of labor and birth .Considered an intervention (the PPA) with high innovative power to promote a new model of care in institutions (the innovation), the main focus of the program was to reduce the high rates of cesarean sections (CS), promote normal birth and qualified care from prenatal to postpartum, through interventions in the care process . Thus, tThe appropriation of a new care model during the adherence decision-making process probably went through people who took into account possible impacts, such as the degree of uncertainty when adhering; the power to solve problems; economic, sustainable, and cultural factors involved in the process, and goals to be achieved after adherence . Other fUsually, innovations pass through a communication transmission channel, in which opinion leaders are potent instruments in the diffusion to their peers, offering greater assurance in the practical use of innovation by the collective during its implementation . Thus, aAccording to Rogers , to makeUnderstanding how hospitals in the private subsystem learned, were motivated and decided to enroll in the Adequate Childbirth Program to innovate their obstetric care model will allow us to understand the limits and achievements in the ongoing process and after implementation. Thus, this article aims to analyze the decision-making process of care managers and leaders to enroll in the PPA. This paper focuses on the pre-innovation conditions and the first three stages of the innovation adoption process described by Rogers Fig. .Recognizing the importance of the Adequate Childbirth Program intervention to the health of Brazilian women and children, the National School of Public Health (ENSP)—Oswaldo Cruz Foundation (FIOCRUZ) proposed an evaluation titled “Healthy Birth: a prospective study to evaluate the implementation and effects of a multifaceted in-hospital intervention.” In order to analyze the implementation and effects of the PPA in a sample of 12 hospitals utilizing mixed methods analysis .The survey was carried out at two different times for data collection. The first [M1] focused on assessing the degree of implementation and the intervention’s effect; the second moment [M2] focused on assessing the sustainability of the intervention. More details on data collection, contextual aspects, and protocols established by the “Healthy Birth” survey can be found in Torres et al.  and DomiIntegrated to this evaluation, this article develops a single case study with an exploratory qualitative approach, which chooses the intervention Projeto Parto Adequado as a case under analysis. For this qualitative analysis, a subsample of 08 (eight) hospitals  was intentionally selected  coaches and remotely for another 02 (two) coaches. In these trainings, there was a reading of the instrument, presentation of techniques for conducting interviews, field observations, and the importance of records.Regarding the characterization of the 04 (four) interviewers who carried out the data collection: they were all women with academic training in Nursing, Midwifery, and History. The historians were researchers in public health; the nurse and the Midwife were researchers in women’s health; all had previous experience with data collection for research. These interviewees were drawn to the field mainly because they were already involved in women’s health research or care practice. They were presented as such to the interviewees.Before entering the field for data collection, pilot tests of the scripts were carried out in a maternity hospital in Rio de Janeiro/Brazil, part of the Adequate Childbirth Project. However, this maternity hospital was excluded from the sample of hospitals in the Nascer Saudável evaluation. This phase made it possible to make adjustments and validate the interview scripts. Furthermore, the research team established a previous contact connection with the management of the institutions to agree on the dates on which the interviewers would be placed in the field for data collecting and any ethical issues that would be released.The immersion in the hospitals took place during a period of 05 (five) days, from July to October 2017, in which the research team carried out interviews with managers coordinators/leaders. A semi-structured script was used for managers and leaders/coordinators, which included the following axes: decision process; deployment strategies; participation of the assistance team; women’s participation; monitoring; strategy results. This script was developed for Healthy Birth research .The interviews took place in the hospitals themselves; privacy was maintained in rooms that could have closed doors and away from other people, with only the interviewer and the interviewee remaining in the room. The interview length (average of forty minutes) was not pre-determined and varied according to each interviewee's level of involvement in the project and subjective features. The interviews were audio-recorded. During this process, there was no need to repeat interviews. Field notes were taken at the end of each hospital field.Subsequently, the interviews were transcribed by an independent professional. Such transcripts were not returned to the participants for evaluation, comments, and corrections. However, for the order of internal validation of the transcripts, there was a review by the research team.About data saturation, it is worth noting that qualitative research, unlike quantitative research, is not based on how many individuals should be heard but on the intensity of the phenomenon and scope to which the actors are linked to the researched object. This emphasizes the need to fine-tune the interviewee selection process. However, the recommended minimum of at least 20–30 interviews was used for qualitative investigations .Work was carried out on constructing and analyzing the database in the MaxQda software . During According to Uwe Flick , the datThe open categorization was then carried out, generating broad segments. With the refinement of these segments, a list of codes was generated, a step called axial coding. An inductive association was made to create the categories . The entAlthough there was no feedback from the participants regarding the findings, for the interpretation of the data, the interviewees’ speeches and their respective coding were validated by members of the research group, in which there were also reflexive co-participations on the interpretive procedures of the entire analytical phase .As a methodological guide for qualitative research, it used the consolidated criteria for reporting qualitative research (COREQ) .The 34 professionals interviewed were directly related to the management and coordination of hospitals, 10 (ten) managers, and 24 leaders/coordinators. These professionals inserted in 08 (eight) hospitals, which distributed in 03 (three) regions of the country  and 06 (six) Brazilian states . Of the 10 managers, 80% (n = 08) are male and 60% (n = 06) are doctors. Regarding the leaders/coordinators, the majority are female 54.2% (n = 13) and 58.3% (n = 14) are physicians .When Brazil, through the hegemonic model of care, produces the cesarean delivery route as an almost predominant form of birth, which does not favor maternal and neonatal morbidity and mortality rates, it is necessary to think of strategies to change this experience.“[…] the Program was presented due to a demand that arose at the time, generated by the Federal Public Ministry. They started a strong campaign and fined the hospitals regarding obstetric violence, and we had the presence, here, of the Federal Public Ministry, and an agreement that the hospital made with the Federal Public Ministry was to be part of the Program […]” (Leader/Coord).One of the responses to the action of the Public Ministry was the proposition, by ANS, of a program to improve the quality of obstetric and neonatal care in the supplementary service through institutional, scientific, and methodological support to hospitals that wished to reorganize concerning prenatal care, delivery and puerperium. The Adequate Childbirth Program fostered the reduction of CS without clinical indications and possible adverse events resulting from inadequate childbirth through the proposition of a new model of care.This situation was the impetus for a feasible and essential shift in obstetric care in the private network, and the ANS’s backing encouraged hospitals to follow the program.In the first stage of innovation, called knowledge, Rogers describes it as “when an individual is exposed to the existence of the innovation and gains some understanding of how it works” . In anal“We received an invitation, directly from the Program Organizing Committee, which is IHI, ANS, and Einstein, inviting the superintendent, our CEO (Chief Executive Office), for a meeting […] where the program, objectives […] would present the program and its objectives” (Manager).The ANS purposefully invited private hospitals with the most significant delivery volume in Brazil1 to an introductory conference where the scope and objectives of the PPA would outline . This me“[…] because you had to apply to view the hospitals that were going to be picked, and they [the two physicians who attended the conference] did the application, and the hospital was chosen.” (leader/Coord)After the meeting to present the PPA, hospitals interested in joining the program registered an adhesion form with ANS, justifying their desire to participate. Soon after, the PPA coordination team would select the hospitals for the program.The desire to change the model of care may or may not have preceded the exposure to the operation of the PPA. However, knowledge of the program through the meeting may have been paramount to raise fundamental questions, such as: how the PPA would work in the maternity ward; how the program would work properly; what would be the costs for hospitals; what would be the support network involved in the change of model, among other issues. According to the “knowledge” stage of the Theory of Diffusion of Innovations , this alThe second stage, called persuasion, is described by Rogers  as when “We used to do our daily routine. Obstetricians who did births introduced news, which we implanted, but it was not a systematic process. That was not the situation.” (Leader/Coord)“It was all the idea of Proper Childbirth. There were isolated ideas , so I thought about some things. I had an idea in the pediatrics, breastfeeding part, other isolated ideas […], but we  did not talk about it (before the PPA).” (leader/Coord).The ANS employed the lack of past interventions or the presence of non-systematized interventions with the potential to change the model of care as a persuasive object for program adherence.Some PPA recommendations, including the participation of management for change, the enhancement of professional, functional performance, the inclusion of women and families in care, and the monitoring of indicators, may have helped the PPA's acceptance. These incitements that, presented during the meeting of presentation of the PPA, may have provided the actors that represented the hospitals and that still did not have structured interventions to change the care model, to adhere to the PPA.“[…] understanding that women currently seek this issue of natural childbirth, of proper childbirth. Furthermore, the hospital has always sought to be within this profile. So I believe that when it went into this field, it has this goal of corresponding to what women today expect from birth.” (leader/Coord).“[…] but as we are in the maternity market, we have every interest in doing the best possible, with all the quality, so, we thought it was important to participate […]” (Manager)The need to belong to representative classes permeates our sociocultural construction. Nevertheless, the social and market status exposed in the speeches reminds us of the need hospitals have in occupying places considered necessary by the social construct, whether by women, families, and social movements or by professionals, scientific academia, and imposing organizations in obstetrics.“When you see the ANS, Einstein, and the IHI, references to quality processes in health care, wow! This group is not kidding. Let us learn! So we had every opportunity, we had a partnership with Einstein, we had open doors at Einstein, our doctors and our nurses participated in the training, realistic simulations, all that there, this is unique.” (leader/Coord).“(…) the Program seemed quite interesting, both from the medical point of view and also, that it adds value to the institution (…)”. (Manager)In this sense, the professionals realized that being aligned to PPA could also open paths for partnerships with other agents involved in the program's composition. Besides the representation of the National Supplementary Health Agency, the professionals pointed out the presence of the Hospital Albert Einstein and the Institute Healthcare Improvement (IHI) as necessary for the adhesion to PPA.Undeniably, to ensure a good quality of care during labor and birth, the physical structure of the maternity ward must be taken into account. Quality of care includes clinical effectiveness, patient safety, and efficiency of care, factors based on indicators such as mortality, morbidity, episiotomy rates, cesarean rates, and use of best practices in labor and birth care.“We had already asked for delivery beds that we had already put in the budget. In short, we were already moinho to be able to attend, in a very timid way, without much ambition […]. I had already visited some other hospitals, and I always brought ideas during my visits. (leader/Coord).“Practically, with the time of one year, before the Adequate Childbirth Program, the hospital started some actions in which we did some small reforms in the pre-birth, to try to leave a little more pleasant and humanized environment.” (leader/Coord).This framework, supported by a practice based on scientific evidence and crossed by individualized, integral, and shared care, ensures the humanization of care. However, it seemed to prevail among managers before participating in the PPA that changes in the physical structure would promote the humanization of care.Before the PPA, the hospitals had already initiated timid modifications related to the structure, which probably favored the decision-making process of adherence to a more systematized intervention program. However, it is impossible to predict if, already in the decision-making process, there was the maturity of understanding of the institutions concerning the physical structure being only one of the components of the scenario in the humanization of care, and not a defining component.“[…] basically our role here, as the leader or head of the service, is that we have a practice in the first place that is safe and a practice that is within the recommendations of the big bodies that govern good practice, among them the World Health Organization.” (Manager).“Not scheduling cesarean sections under thirty-nine weeks, we had already started in the year 2014, because we had already seen by the indicators that we had a high rate of respiratory distress (in newborns), by obstetricians performing cesarean sections in those children that are considered late preterm.” (Manager).“Basically we tried to do prenatal care within the best standards of protocols, bring some degree of preparation to these patients, through courses for pregnant women […]” (leader/Coord)“[…] we needed to change obstetrics in Brazil, especially in private health, because it had become a cesarean factory and that we had abandoned traditional obstetric practices, and normal became cesarean.” (Manager)“[…] our kind of service had been facing difficulties concerning this epidemic of cesarean sections […]we were not knowing, by ourselves, how to dribble these difficulties.” (leader/Coord)This category was defined by reports relating to efforts that the hospital had begun to execute before the PPA but had not progressed and was aided by the program Fig. . In addi“So that is how it happened, so I brought (agents who influenced the decision to join the PPA), I made a report […], and he (the decision agent) understood that we should participate in the program and then, we started.” (Manager).“[…] I had a series of meetings, I showed the Program, I talked to one, I talked to another, until we received the endorsement, the release, from the [decision-making agent], to participate in this Program.” (leader/Coord).The perceived characteristics of the innovation are put to the test when hospital representatives attend the program presentation meeting and return with their relative advantages and possible disadvantages of adopting PPA. This internal “evaluation” process of the hospitals considered compatibility, desire for change, complexity in implementing such an intervention, the perception of women, and health plans in enjoying an interventional innovation, among others.For this study, we consider as opinion leaders, here represented by influencing agents, those who presented a favorable/disfavorable attitude to the hospital administration regarding adherence to the PPA; as well as change agents, symbolized as decision making agents, actors responsible for the final decision and for signing the term of adherence to the program. Nothing prevents the same person from assuming the two positions; what depends on the degree of management involvement regarding innovation.Over the years, several normative , 23 and Thus, when the PPA comes up with a proposal to change the obstetric care model in private hospitals, it suggests an intersection between health and innovation. Such an intersection is challenged not only by the need to change poor indicators but also by the entrepreneurial vision of the very leaders involved in the adoption of PPA, who must adjust to the needs and expectations of patients, organization professionals, and, most importantly, the institutional mission’s yearning .In knowing about the adherence to innovation, it should be apparent to those involved that the proposed innovation is not a neutral act of people or institutions because it needs to be based on its possible effects to implement it. Thus, identifying the need for the innovation; in which body such innovation will be implemented; whether it will be socially constructed; whether it maintains minimum conditions of access ; in addiOne should be careful about the possible adverse impacts when applying innovation in health institutions since it does not necessarily have only positive consequences . Recent Only the strengthening and empowerment of women is ineffective when the cesarean section has become normalized in Brazil by the thinking and attitude of specialists, which is perpetuated in health institutions as something simple, safe, aesthetic, clean, and painless , 29. WomThere is, then, a real need for the strengthening and recognition of the intentions that surround private obstetric health institutions in Brazil, conformed by a mission, vision, and philosophy that must dialogue with the wishes of women, with the best scientific evidence, and with the humanization of labor and birth. By joining the PPA, the proposal to change the care model seemed to go beyond building a new professional way of acting, punctuated only by specific training, but also to a follow-up of the changes over time, to the recognition of people strategies that could encourage changes in conduct, practice, and care, envisioning a new institutional and not only professional thinking and positioning .In this sense, finding strategic leaders , 34 for However, it was noticed that the choice of this change agent in charge of sustaining the intervention was influenced not only by his professional knowledge and technical abilities but also by his institutional influence over the remainder of his category and other professional categories , 37. TheThe adherence to the Adequate Childbirth Program went through the appropriation of the innovative intervention to then adopt it. The professionals representing the institutions, during this decision-making process, felt privileged to have received exclusive invitations to participate, sent by the organizers of the program. This simple act expresses special care, identifying each service as unique.The lack of multifaceted obstetric interventions in the Brazilian private service, the social and market status, the search for better care, and the possibility for hospitals to carry out structural reforms by being inserted in the intervention motivated adherence to the PPA. The use of agents of influence to convince peers was key to the final decision to join.The speech place of the persons interviewed is an essential factor to consider since it might limit the facts in their natural context to match the research institution’s expectations. Furthermore, the data was obtained after the participants had joined the program, favoring possible memory biases during data collection. Finally, there was a gap between the time the data was collected and its analysis. Our research, however, revealed the logic of a work shaped by institutional, social, and cultural rules that do not change quickly, that require time, desire for change, and training to improve care, which reinforces the validity of this study.Journals rarely publish the point of view of management and coordination professionals in the innovation decision-making process. The focus often permeates the deployment results. In this research, we have the opportunity to deal with what motivated midwifery managers and leaders/coordinators in the Brazilian private sector to seek a change in the care model.This fact reveals that to continue reducing cesarean sections in complementary institutions in Brazil, complex interventions, such as the PPA, are needed to ensure better results. Another fact revealed by this study permeates the transmission of knowledge about PPA, having occurred strategically by members of institutions considered socially representative and respected in the professional field, in this case, the physician. This may allow a greater adherence of professionals. However, we still do not know if this fact revealed greater assurance in the implementation and sustainability of the program in these institutions.


Data seem to suggest that NMP can enhance the preservation of liver transplantation to some extent and improve the early outcome after transplantation. In this review, we provided an overview of NMP and its application in ex vivo liver preservation and pre-transplantation, and we summarized the data from current clinical trials of normothermic liver perfusion.The discrepancy between the number of patients awaiting liver transplantation and the number of available donors has become a key issue in the transplant setting. There is a limited access to liver transplantation, as a result, it is increasingly dependent on the use of extended criteria donors (ECD) to increase the organ donor pool and address rising demand. However, there are still many unknown risks associated with the use of ECD, among which preservation before liver transplantation is important in determining whether patients would experience complications survive after liver transplantation. In contrast to traditional static cold preservation of donor livers, normothermic machine perfusion (NMP) may reduce preservation injury, improve graft viability, and potentially  Patients with liver cirrhosis, decompensated illnesses, acute liver failure, hepatocellular carcinoma, and other irreversible liver disorders are currently treated with liver transplantation as a first choice . Howeverin vitro, NMP provides oxygen and nutrition at 37°C, which could decrease the risk associated with marginal liver transplantation   . In conty death) . Machinein vitro is the most crucial link in this chain. Organ transplantation research is challenging because once the organ is removed, the original blood circulation ceases instantly. The first goal of organ perfusion was to maximally eliminate toxic metabolites (+ channel (ENaC) activity, which leads to the dysfunction of ENaC and the decrease of renal function  perfusate . CellulaAt present, the effective artificial oxygen transporters are hemoglobin based oxygen carrier (HBOC). Many oxygen carriers are used in clinical and NMP, including Hemoglobin Vesicles , Hemogloex vivo organs is the key point in organ transplantation  improved efficacy in providing prolonged hepatic oxygenation, 2) was able to provide normal hepatic functions  over a 12-h period, 3) normal anatomical hepatic features after 12-h of ex vivo preservation, 4) mitochondrial function was sustained within normal range during the 12-h ex vivo period, 5) the magnitude of NO oxidation (nitrite and nitrate levels) was lower compared to previous published data, and 6) hepatocytes sustained full cellular integrity over the 12-h period, including normal anatomical features for mitochondria. Taken together, these parameters indicate that the VIR-XVl maintained intact and fully functional during the 12-h study.To address the urgent medical need in liver transplantation, VirTech Bio, Inc. (VTB) has explored using a non-cellular HBOC to address the concept that effective oxygenation of isolated antation . Thus, VOxyVita is a new generation of HBOC. The OxyVita has been shown to be extremely resistant to breakdown and unfolding, resulting in a molecule that not only starts out as a large polymer, but should stay as such after injection. The zero-link polymerization technology, at its core, is based on removing any residual linking agents within the polymer itself. This eliminates concerns of reverse reactions or decomposition due to non-specific binding, temperature and pressure changes also eliminate the risks of residual linker toxicity.2 pressure between 9 mm Hg and 30 mm Hg. They do not contain clinically relevant RBC antigens and have a longer shelf life than RBCs. The porcine models of using Hb-Vs for perfusion (2 consumption during subnormothermic machine perfusion (SNMP) and decreased alanine aminotransferase and lactate dehydrogenase levels after reperfusion compared to hypothermic machine perfusion (HMP) and SNMP, without an additional OC.Hemoglobin-vesicles (Hb-Vs) are phospholipid vesicles containing human-derived Hb. The diameter of the vesicles is 250–280 nm, which is smaller than that of RBCs. Hb-Vs are saturated for 50% at an Oerfusion  showed i2life was the first HBOC recently approved by the European Health authorities. The natural extracellular Hb equivalent Hemarina M101  is obtained from a marine invertebrate: Arenicola marina, a lugworm. It is composed of 156 globins and 44 non-globin linker chains that can carry up to 156 O2 molecules when saturated, which results in a high O2-binding capacity. Hemarina M101 is active over a large range of temperatures (4°C to 37°C) and releases O2 according to a simple gradient that does not require any allosteric effector. The molecule possesses intrinsic Cu/Zn-superoxide dismutase antioxidant activity that, to a certain extent, protects tissue from superoxide radicals  and has shown superior results in organ preservation. In an obese Zucker rat model  and lactate levels at the end of reperfusion were significantly lower in the group preserved with M101 (p < 0.05). Protection from reactive oxygen species  and higher production of NO2-NO3) and less inflammation (HMGB1) were also observed in this group (p < 0.05). These data demonstrate, for the first time, that the addition of HEMO2life® to the preservation solution significantly protects steatotic livers during SCS by decreasing reperfusion injury and improving graft function. In addition, M101 the only Oxygen Carrier (OC) compatible for a wide range of temperatures, suggesting that it can be used in hypothermia as well as normothermia.HEMOradicals . HEMO2liat model  et al., The history of NMP began with the development of a perfusion chamber for organ preservation at room temperature in 1935 by Alexis Carell and Charles Lindbergh . HoweverClinical findings show that the perfused liver in the first phase clinical trial of NMP  has stabin situ normothermic regional perfusion (NRP) with normothermic mechanical perfusion, the By combining Around two billion individuals worldwide have hepatitis B, and one million of them pass away from the disease late consequences, according to WHO figures . Since tThe risk assessment of whether an isolated liver meets the criteria for transplantation has not been established until now. Liver donor criteria usually include age, recipient criteria, and gross appearance of the liver. The subjective, unstandardized assessment of the surgeon is an important reference for the acceptability of the donor liver. The lack of objective, independent predictors and variables to predict graft function after transplantation leads to the discarding of potentially functional livers. In the clinical practice of NMP, such as elderly donor liver or steatosis liver with DCD can get objective evaluation, which has a positive effect on whether more donor liver can be applied in clinical transplantation . NMP wasex vivo liver perfusion (NMP-L). Evaluation protocols included perfusate lactate, bile production, vascular flow, and liver appearance. Before NMP, all the livers were exposed to static refrigeration at different periods, and the grafts played an immediate role after transplantation. During an average follow-up of 7 months (ranging from 6 to 19 months), liver function tests were normal. In this study, the researchers successfully evaluated and resuscitated a batch of “rejected” liver transplant patients by testing the viability of some high-risk donor livers during NMP-L, and the patients recovered after transplanting these high-risk donor livers. This pilot study shows that a proportion of high-risk donor livers might be transplanted by subjecting them to viability testing during NMP-L, without compromising patient safety in a cohort of low-risk recipients.p = 0.001). The researchers also concluded that prolonged normothermic preservation might allow time for recovery, and it might also offer a useful platform for therapeutic delivery (steatosis reduction). NMP allows functional parameters to be measured as a marker of the viability of the organ: prolonged NMP might allow clinicians to accept a high-risk organ on the basis .Thomas According to the previous research reports, although there is no completely determined standard for the evaluation of liver function by NMP before liver transplantation, the currently widely accepted evaluation standards  include:ex vivo normothermic machine perfusion. In this Phase 1 trial, NMP livers were matched 1:2 to cold-stored livers. Twenty patients underwent liver transplantation after NMP. This first report of liver transplantation using NMP-preserved livers demonstrates the safety and feasibility of using this technology from retrieval to transplantation, including transportation. NMP may be valuable in increasing the number of donor livers and improving the function of transplantable organs. The primary endpoint was 30-day graft survival. The results showed the procedure is feasible and safe. All livers enrolled into the study were successfully transplanted with 100% 30-day recipient and graft survival. This is the first report of normothermic perfusion in clinical liver transplantation. This preservation methodology potentially reduces the risk inherent in transplanting marginal donor organs. The researchers believe that normothermic preservation may substantially improve organ utilization.According to a report of Watson  on a 57-In a pilot, open, randomized, prospective trial, p = 0.03). During the 6-month follow-up, none of the patients receiving NMP transplantation developed ischemic cholangiopathy (IC). The purpose of this study is to explore whether liver transplantation can be delivered from the donor center of SCS until NMP was started at the recipient center, because this will simplify logistics and reduce the cost of the transplantation process. In fact, the results of this study indicate that NMP after SCS can be safely used for low-risk DCD and DBD grafts.ex vivo assessment capability of donor allografts, extent of reperfusion syndrome, incidence of IBC at 6 and 12 months, and overall recipient survival after transplant. Among the per-protocol patients (293 patients), 151 were in the OCS-liver group and 142 were in the ICS group. The primary effectiveness end point was met by a significant decrease in EAD . The OCS Liver preserved livers had significant reduction in histopathologic evidence of ischemia-reperfusion injury after reperfusion . The OCS Liver was also associated with significant reduction in incidence of IBC 6 months  and 12 months  after transplant. This multicenter randomized clinical trial provides the first indication, that normothermic machine perfusion preservation of deceased donor livers reduces both posttransplant EAD and IBC. Use of the OCS Liver also resulted in increased use of livers from donors after cardiac death. Together these findings indicate that OCS Liver preservation is associated with superior posttransplant outcomes and increased donor liver use.For the three years from November 2016 to October 2019, 1. NMP can test the safety and feasibility of transplant donors before transplantation.2. NMP can dynamically evaluate liver transplantation.3. NMP may detect bile duct injury caused by IRI earlier than other clinical variables.4. NMP helps to reduce macrovascular hepatic steatosis.1. The liver active markers during NMP perfusion should be standardized, which is the key to promote safely and rapidly this field.2. Physiological mechanism of NMP activates liver donors and optimizes existing technologies.ex vivo transplantation, and a retrospective analysis of the current trails on the preservation and transplantation of human liver by normothermic machine perfusion. The feasibility evaluation study of NMP applied to liver transplantation shows that objective and definite criteria for liver function indexes of liver transplantation are needed to ensure that the donor liver can be successfully transplanted into a patient. NMP-L provides survival parameters of liver function before marginal donor liver transplantation, which can increase organ utilization and reduce patient mortality. The use of NMP-L in liver transplantation and preservation shows that it has such evaluation function and has certain feasibility. NMP has advantages in increasing the utilization rate of marginal liver and alleviating the shortage of donor livers for liver transplantation. Future double-blinded, randomized, large clinical trials will be required to evaluate the benefits of NMP technology.The purpose of this review is to provide an overview of NMP and its application in the preservation of liver before


These data indicate that R-carvedilol could be a potential new therapy for AD.For decades, the amyloid cascade hypothesis has been the leading hypothesis in studying Alzheimer’s disease (AD) pathology and drug development. However, a growing body of evidence indicates that simply removing amyloid plaques may not significantly affect AD progression. Alternatively, it has been proposed that AD progression is driven by increased neuronal excitability. Consistent with this alternative hypothesis, recent studies showed that pharmacologically limiting ryanodine receptor 2 (RyR2) open time with the R-carvedilol enantiomer prevented and reversed neuronal hyperactivity, memory impairment, and neuron loss in AD mouse models without affecting the accumulation of  In addioid (Aβ) . Unfortuoid (Aβ) . These d+/+ mouse model, Given the multifactorial nature of AD pathogenesis, multiple pathways besides Aβ metabolism could be targeted for AD treatment. Indeed, recent studies have revealed many new targets and strategies for treating AD. For example, a molecule named leuco-methylthioninium bis(hydromethanesulphonate) (LMTM) that is thought to block the aggregation of tau has been shown to possess some promising anti-AD properties in a phase III clinical trial . FurtherIn addition to these new trends in AD treatment, substantial evidence indicates that soluble Aβ induces neuronal hyperactivity, which in turn generates more soluble Aβ. These two components form a vicious cycle . This Aβß-adrenergic receptor blocker, widely used for the treatment of congestive heart failure  in mouse models of familial AD (FAD) in vivo and in vitro (Carvedilol is a nonselective  failure . A previerfusion . However-limited . The clirvedilol . Recentlin vitro . This miα-blocker) and S-carvedilol (an α- and β-blocker). In vitro studies have shown that S-carvedilol has approximately 100-fold greater affinity for adrenergic ß-receptors than R-carvedilol. Meanwhile, R-carvedilol and S-carvedilol inhibit adrenergic α-receptors to the same extent  associated with catecholaminergic polymorphic ventricular tachycardia (CPVT). Importantly, RyR2-R4496C+/- mice that received R-carvedilol treatment did not show a significant change in heart rate or blood pressure . Single channel recordings revealed that carvedilol can directly shorten the open time of the ryanodine receptor 2 (RyR2) Ca channel . Like thpressure .ß-blockers, carvedilol is a far more potent antioxidant. The antioxidant effect of carvedilol mainly resides in the carbazole moiety, and the substitution of a hydroxyl group at certain positions on the phenyl ring of either carbazole or the ortho-substituted phenoxylethylamine part of carvedilol resulted in an increase in antioxidant activity  littermates. Notably, compared to DMSO-treated control group, pre-treatment with R-carvedilol for one-month, markedly reduced caffeine-induced calcium release in CA1 pyramidal neurons from acute 5xFAD+/- mice brain slices  and 3–4 months old    were pre in vivo . Similarl slices . Thus, b+/- mice (before AD symptoms) prevented memory loss and reversed LTP deficit, while in 3–4 months old 5xFAD+/- mice (after AD symptoms), pretreatment with R-carvedilol also reversed these deficits. The drug was also tested on 6–7 and 10–12-month-old 5xFAD+/- mice to further assess whether R-carvedilol pretreatment remains effective in advanced AD stages. Results from behavioral tests and LTP recordings also suggested that pretreatment with R-carvedilol could still reverse memory deficits even in 6–7 months and 10–12 months old 5xFAD+/- mice .  test and novel object recognition (NOR) test, together with measurement of hippocampal CA3-CA1 long-term potentiation (LTP). Similar to the results of calcium imaging, pretreatment with R-carvedilol in 2–3 months old 5xFADate AD). .+/- mice . The exact reason for the ineffectiveness of the carvedilol racemic mixture in inhibiting AD progression is unknown. One possibility is that the strong ß-blocking effect of S-carvedilol in the racemic mixtures, especially at high doses, may adversely affect neuronal activity and cognitive function, thus counteracting the beneficial effects of R-carvedilol. Furthermore, the beneficial effect of R-carvedilol on learning and memory is dose-dependent. R-carvedilol at a dose of 3.2 mg/kg/day or 1.6 mg/kg/day, but not 0.8 mg/kg/day, rescued cognitive decline in 5xFAD mice.As mentioned above, previous studies suggested that the racemic carvedilol may reduce the expression of oligomeric Aβ and prevent plaque formation and cognitive decline in multiple mouse models of AD . However+/- mice . This ne+/- mice (12–15 months old) were treated with R-carvedilol or DMSO for one month and behavioral tests and LTP measurements were conducted. Similar to the studies with 5xFAD mice, +/- mice pretreated with DMSO, 3xTG+/- mice with R-carvedilol pre-treatment spent significantly less time to find the target platform. While in the probe trials, 3xTG+/- mice that received R-carvedilol pre-treatment spent significantly more time in the area where the platform used to be. Interestingly, mice received R-carvedilol pre-treatment also showed increased speed of swimming compared to the DMSO control group. Similarly, results from the NOR test suggested R-carvedilol pre-treatment increased the discrimination index and the walking velocity in the 3xTG+/- mice. LTP recordings also suggested that R-carvedilol pre-treatment significantly improved the neuronal circuit function in 3xTG+/- mice (AppNL−G-F mouse model, the AppNL/NL and AppNL−F/NL−F mouse models, and the APOEε4+/+ mouse model (In the study of +/- mice . In lighse model .+/- and 3xTG+/- brain slices (+/- and 3xTG+/- mice showed a significantly higher density of neurons in the subiculum area compared to the DMSO-pretreated mice. These results suggested that, like carvedilol, R-carvedilol may also have the ability to protect neurons from neuronal cell death. Besides neuronal hyperactivity, learning and memory impairment, and neuronal cell death, accumulation of aggregated Aβ in the brain is believed to be one of the major hallmarks of AD (+/- mice or 3xTG+/- mice indicated that R-carvedilol pre-treatment did not affect Aβ accumulation (Neuronal cell death is one of the clinicopathological features observed from human AD patients , but neun slices . Notablyks of AD . R-carvemulation . TherefoCarvedilol has been used for treating cardiovascular diseases for decades. New indications for carvedilol are emerging. In addition to its beneficial effects on the heart, carvedilol has been shown to have potential for combatting cancer, neurological disorders, and other diseases . The lis


Educating the at-risk population about stroke symptoms and requirement of calling an ambulance when stroke strikes is challenging. This exploratory cross-country study provides insights to the FAST Heroes educational campaign and outcomes hitherto achieved.The primary aim of the study was to measure the transfer of stroke-related knowledge to parents after a global school-based FAST Heroes educational campaign for 5- to 9-year-old children in 14 different countries. The secondary aim was to evaluate parents and teachers' acceptability toward the program.The duration of the program was 5 h; 1 h per week, joining face-to-face educational sessions with workbooks, cartoons, web-based learning, and other fun activities. Outcomes were measured before implementation (t1), after implementation (t2), and at 6-month follow-up (t3). Program acceptability and stroke knowledge were evaluated by feedback surveys for teachers and parents.p < 0.001). All three surveys were completed by 86 parents, who improved their knowledge of stroke symptoms, 55% (t1), 79% (t2), and 94% (t3) (p < 0.001). Overall, the educational messages were successfully passed onward.Worldwide, 4,202 parents completed the program with their children and answered surveys at t1 and t2. They increased their knowledge of three stroke symptoms from 48 to 83% (Findings confirm the primary aim of the study that is knowledge about stroke transfer well from children to their families through the FAST Heroes program. Second, parents and teachers globally consider the program feasible and worthwhile. The results will inform further rollout of the campaign. Stroke is the second leading cause of death worldwide and the leading cause of acquired disability in Europe . The numTo understand how to educate the public about stroke, one first needs to understand whom to educate. The median adjusted age at which the first stroke occurs is 70.3 years in Europe (with an interquartile range of 14.1 years) , 17. ConOver the last two decades, several health-related campaigns targeting children have shown impressive results –27. The The FAST acronym was developed in the UK in 1998 and has been culturally adapted worldwide. Studies using FAST have demonstrated high accuracy in diagnosing strokes by paramedics and emergency medical technicians with positive predictive values between 61 and 77% . The “GrThe primary aim of this descriptive cross-country study is to disseminate the first results of the global “Grand Mission” FAST Heroes program and its effectiveness on knowledge transfer to parents of participating children worldwide. Researchers have recently referred to “stroke knowledge” as knowledge of stroke warning signs, and appropriate behavioral response to stroke symptoms , 34. In The FAST Heroes campaign includes a comprehensive set of educational resources that was designed and developed in 2018 by a multidisciplinary team consisting of kindergarten teachers, health care professionals, school psychologists etc. A series of pilot studies were used to assess children's baseline stroke knowledge and fine-tune the educational approach for 5- to 9-year-old children so they carry their stroke-related knowledge onwards to their parents For further details please see , 32, 35.The successful implementation of the program on a national level in Greece justified its expansion globally. To date, the campaign has been adapted to 11 local emergency numbers, i.e., 103, 106, 107, 112, 123, 131, 192, 911, 995, 998, 999. It has also been translated into the local language for a still growing list of more than 37 communities, including Argentina, Belarus, Brazil, Bulgaria, Canada, Catalonia, Chile, Colombia, Croatia, Czech Republic, Ecuador, Egypt, Georgia, Germany, Greece, Hungary, Iceland, Italy, Iran, Kazakhstan, Lithuania, Malaysia, Moldova, Peru, Poland, Portugal, Romania, Russia, Singapore, Slovakia, South Africa, Spain, Ukraine, United Arab Emirates, United Kingdom, and Uzbekistan. From March to June 2021, a global initiative called “Grand Mission” was launched, through which we aim to enroll one million children and their families worldwide in the next 5 years . Public The project received endorsement of the World Stroke Organization , Stroke Alliance for Europe , and the Schools for Health in Europe Network Foundation . This study received clearance from the Committee for Research Ethics of the University of Macedonia  (14/15.06.2020), where the program's educational content was developed. The ethical permission is in accordance with the 1964 Declaration of Helsinki.Three assessment tools were used in the present study in total . Parentsvia email to complete it voluntarily. The PFSt and PFSp survey consisted of 27 and 28 questions, respectively  and Project Feedback Survey for Parents (PFSp) and participants were directed toward it ectively  and hostAll parents who had registered on the website with their children participated in the SPQ assessment. Due to different starting dates of the program in each country, parents were divided into 2 groups: Parental group 1 consists of parents who completed the two assessments of the SPQ (t1 and t2), group 2 consists of parents who completed the SPQ in all three assessments . Teachers and parents from Greece, Poland, Romania, Slovakia, and Ukraine, which were the countries with the most schools participating in the program, were targeted to measure their level of satisfaction and acceptance of the campaign.IBM SPSS Statistics 25 software was used. The McNemar test for related samples examined the relation of parents' answers in t2 and t3 as regards stroke symptoms  and the selected course of action in the event of witnessing a stroke . A Chi-Square test examined whether the educational program impacted parents' knowledge in relation to choosing the corresponding emergency number in each country in t2 and t3 . All tests were conducted at the α = 5% significance level.n = 4,202). At t1, 32% of parents chose “Call an ambulance” for all three symptoms vs. 70% at t2. Almost half of the parents (48%) were able to identify all three stroke symptoms at t1 vs. 83% at t2 . A Chi-Square test showed that the educational program had a significant effect regarding the emergency number respondents would select to call an ambulance (question 3) (p <0.001).n = 86 parents). At t1, 30% of parents chose the correct response for all three symptoms, 65% (t2) and to 83% (t3) (p < 0.001). Parental group 2 comprises parents who completed the program and assessments at t1, t2, and t3 (83% (t3) . More th83% (t3) . All parSupporting an important cause that can make a positive difference” was selected by 85% of teachers for participating in the program, 64% of teachers selected “It can help spread an important message”, and 40% selected “It looked interesting and rewarding for children”. Some teachers (38.4%) knew a close relative or family friend who had suffered a stroke. Also, teachers agreed or strongly agreed that the campaign could help save lives (97.3%), that knowledge transfer had taken place (93.4%), that the children enjoyed taking part in the campaign (97.4%), and that the materials were of high quality (96%). “The characters/FAST Heroes” ranked the highest, followed by “the animated films”, “the way they make learning fun”, “the online e-books” and “the educational workbooks”. Almost the two thirds (62.5%) of teachers received unsolicited positive feedback from parents. The vast majority (98.1%) agreed or strongly agreed that they would recommend the campaign to others and 98.6% reported that the campaign had increased their confidence about what to do if somebody had a stroke. PFS was completed by 675 teachers and 417 parents from Poland, Slovakia, Romania, Greece, and Ukraine. In regard to the teachers, 37.5% of them implemented the educational program with 11–20 children, while 33.2% of them implemented the program in classes of 21–30 children. Schools were mainly located in towns or cities with fewer than 20,000 inhabitants (41.2%). The program was presented face-to-face by 59% of the teachers, whereas 224 teachers (33.2%) used a hybrid model and 53 teachers (7.8%) implemented all modules virtually. “Sixty one percent of the children in the survey were 5–9 years old and 43% were >9 years old. Thirty one percent of parents in our survey had parents or parents-in-law living with them and 38.1% had a close relative or family friend who had suffered a stroke. Parents that agreed or strongly agreed that their children enjoyed taking part in the campaign (94.5%), that their child understood the message (93%), that their children had passed on the messages to their family members (86.8%). Parents agreed or strongly agreed that the campaign increased their own confidence about what to do when somebody had a stroke (91.4%), that the campaign could help save lives (93.7%), and that they would recommend the campaign to others (91.8%).Process and outcome evaluations of the global FAST Heroes campaign were explored in this study in conjunction with the effectiveness of strategies used for its development and implementation. We examined the extent to which the campaign succeeded to meet its primary objective, that was evaluating the program's impact on knowledge delivery from children to their parents, as well as its secondary objectives, which included reporting parents' and teachers' perceptions about the campaign.Results from parental group 1 who completed the SPQ in t1 and t2 of the study showed that they chose “Call an ambulance” for all three stroke symptoms in t2 which was a significant increase. Marto et al.  had simiStroke literacy of parents has been examined up to 3 months post-intervention . KnowledTeachers from 5 European countries  participated in the current study. Nearly all teachers (98.1%) would recommend the campaign to others. Almost half of the teachers delivered parts of the program remotely. Due to local lockdown restrictions during the COVID-19 pandemic, almost half of the teachers delivered parts of the program remotely using online learning tools. This suggests that the program can be implemented both in class and digitally and may thus be viable to implement even in remote areas. Most teachers (90%) also recognized that children had fun while participating and that the educational material contained entertaining ways of transferring knowledge between children and their families.Altogether, results concur with the primary goal of the FAST Heroes campaign, which is to educate young children and their extended families to recognize the main stroke symptoms and act urgently in the event of a stroke.The descriptive exploratory nature of the study made it impossible to provide firm conclusions regarding causality. A recent systematic review and meta-analysis of community stroke educational programs found only two randomized studies in the literature . Strict Other factors  may have increased parents' stroke-related knowledge independent of the program. We cannot control parents' access to knowledge but consider this a welcomed facilitating ripple effect. Additionally, the SPQ is a variation of the Stroke Action Test (STAT)  and is nFuture studies should measure the campaign's impact on the percentage of stroke patients who arrive at the hospital within the time frame to receive medical emergency treatment and changes in the delay of hospital arrival after implementing the FAST Heroes program. Furthermore, focusing on relatives' first reactions and mechanisms behind improving stroke-related knowledge could be obtained by incorporating qualitative questions into evaluations.Our results demonstrate that the implementation of “Grand Mission” FAST Heroes educational program for 5- to 9-year-old children, which is nested in an ongoing global campaign, has already yielded positive results in the knowledge of thousands of families in different settings and countries. Findings confirm that knowledge about stroke transfers well from children to their families through the FAST Heroes program despite regional differences. A novel result of this study is the affirmed trend toward knowledge retention of parents 6 months after completion of the program. Importantly, parents and teachers consider the program feasible and worthwhile in a wide variety of settings. The results will inform the further rollout of the campaign and future studies. Findings are also likely to be useful in our long-term objective: to get FAST Heroes included in school curriculums to ensure continuous tuition of essential stroke symptoms to families.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by Committee for Research Ethics of the University of Macedonia  (14/15.06.2020). Written informed consent to participate in this study was provided by the participants' legal guardian/next of kin.All authors listed have made a substantial, direct, and intellectual contribution to the work and approved it for publication.This study received funding from Boehringer Ingelheim . The funder was not involved in the study design, collection, analysis, interpretation of data, the writing of this article, or the decision to submit it for publication.KT, JM, CW, and HP are employed by Boehringer Ingelheim. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Combining adaptive and innate immunity induction modes, the repertoire of immunoglobulin M (IgM) can reflect changes in the internal environment including malignancies. Previously, it was shown that a mimotope library reflecting the public IgM repertoire of healthy donors (IgM IgOme) can be mined for efficient probes of tumor biomarker antibody reactivities. To better explore the interpretability of this approach for IgM, solid tumor-related profiles of IgM reactivities to linear epitopes of actual tumor antigens and viral epitopes were studied. The probes were designed as oriented planar microarrays of 4526 peptide sequences (as overlapping 15-mers) derived from 24 tumor-associated antigens and 209 cancer-related B cell epitopes from 30 viral antigens. The IgM reactivity in sera from 21 patients with glioblastoma multiforme, brain metastases of other tumors, and non-tumor-bearing neurosurgery patients was thus probed in a proof-of-principle study. A graph representation of the binding data was developed, which mapped the cross-reactivity of the mixture of IgM (poly)specificities, delineating different antibody footprints in the features of the graph—neighborhoods and cliques. The reactivity graph mapped the major features of the IgM repertoire such as the magnitude of the reactivity (titer) and major cross-reactivities, which correlated with blood group reactivity, non-self recognition, and even idiotypic specificities. A correlation between an aspect of this image of the IgM IgOme, namely, small cliques reflecting rare self-reactivities and the capacity of subsets of the epitopes to separate the diagnostic groups studied was found. In this way, the graph representation helped the feature selection in its filtering step and provided reduced feature sets, which, after recursive feature elimination, produced a classifier containing 51 peptide reactivities separating the three diagnostic groups with an unexpected efficiency. Thus, IgM IgOme approaches to repertoire studies is greatly augmented when self/viral antigens are used and the data are represented as a reactivity graph. This approach is most general, and if it is applicable to tumors in immunologically privileged sites, it can be applied to any solid tumors, for instance, breast or lung cancer. Any trivial antibody test is used like a keyword search of a log of pathogen encounters. The whole repertoire of antibodies, on the other hand, contains far more information. Attempts to read the repertoire rather as a map of immunological meaning are still rare. The structural studies using antibody repertoire sequencing ,2,3 haveThe IgM repertoire is a pool of immunochemically largely indistinguishable antibodies that are the product of several B cell compartments . They caThe immune system detects tumor-associated antigens (TAA) and appropriately reading the imprint of this information, e.g., in the repertoire changes, could be a source of clinically relevant information ,18,19. S®, Heidelberg, Germany). It contains a sequence set derived from 20 whole-length human proteins known to be TAA as well as 209 viral linear B cell epitopes related to malignancies. Confirming the expected low interpretability of IgM reactivities with self-antigens in the terms of adaptive responses, our results nevertheless identify patterns of IgM reactivity, which generally classify brain tumors. The interpretability of this differential IgM reactivity was found in terms of classes of polyspecificity, mapped using a reactivity graph, and perturbations of the IgM antibody levels.With these considerations in view, we previously argued that the repertoire of IgM may be an untapped source of biomarkers ,26. It wH. pylori) related to cancer , cerebral metastases  or control (n = 6). Control subjects were selected randomly among cases with degenerative spine disease. Patients who had undergone previous radio-, chemo- or immunotherapy, who suffered from an autoimmune disease or who had an inflammatory disease in the past three months were excluded. After staining, reading, cleaning, and normalizing for both binding dependence on the amino acid composition  and for chip effects , the data represented the IgM reactivities of 21 patients with 4512 peptides. The latter included 24 human tumor antigens scanned with a 15 amino acid frame shifted by two positions and the known linear B cell epitopes from 29 protein antigens of pathogens, mostly viruses  peptide sequences provided a control group of putatively cross-reactive peptide pairs. The Euclidian distance, correlation or cosine distance was considered lossy or inadequate. The ANOVA F statistic proved more informative because it is independent of the global mean level of the compared pair of profiles but is affected by the difference in the mean levels of the pair. Therefore, it was adopted as a similarity measure although it is not a true metric.The reactivity profiles of a pair of peptides with a set of N sera may be represented as a comparison between N groups, each of two points. The F statistic is the ratio between (a) the variance of the means of the groups and (b) the mean of the variances within the groups. Thus, when two profiles are similar, the two points in each group are close while the overall reactivity differs much more between groups . To measThe graph, thus constructed, had 4512 vertices and 77,914 edges. The diameter of the graph—the shortest path between the farthest vertices without edge weights, was 12. The graph consisted of a large connected component of 4455 peptides and 52 disconnected peptides as well as three pairs unrelated to the rest. The vertex degree (number of neighbors) had an exponential distribution, which is a property of networks evolving by random attachment  28]. Ta. Ta28]. This visualization is a provisional embedding for illustrative purpose, so the dimensions are not intended to be interpretable. Nevertheless, the form and the relationship between the clusters of vertices are interpretable. The long horizontal axis along which the vertices are spread correlated with the mean intensity of the IgM reactivity in the profiles , but theThe layout of the graph was constrained to two dimensions, and in view of its high connectivity, significant topological information could be lost. Indeed, a hierarchical recursive Louvain clustering and subsequent optimal tree cutting yielded 15 clusters (communities) with higher edge density within than between the clusters A. FourteThe highly connected graph contained 239,349 maximal cliques of 3 to 38 vertices . A cliquhttps://meme-suite.org/, accessed on 13 December 2022 [p < 0.05). The k-mer distance between the sequences showed an increase of only 1% in similar sequences in the large cliques . Therefore, it was hypothesized that the large cliques are not due to degenerate epitopes but most probably to abundant oligoclonal public reactivities.A number of motifs were extracted from the set of peptides found in the cliques of 20 sequences and larger using the XSTREME algorithm from the MEME suit (ber 2022 ), and thThe coincidence of the clusters of high density containing the large cliques and the parts of the graph with apparently divergent anti-A and anti-B reactivity  suggesteThe dichotomous separation of cross-reactivities associated with blood group antigen expression covered a wide and diverse set of peptides and was proven to be among the major factors defining the topology of the graph being related to dense clusters and large cliques.Another possible source of highly expressed public IgM reactivities, testable by the set of antigens used, was antiviral responses, although this would be more relevant in the case of IgG and IgA antibodies. More viral linear epitopes than self-epitopes participated in the large cliques (>20 vertices), and the opposite was true for the small cliques (<5 vertices) . This efInterestingly, the most prominent viral specificities were several HTLV1 epitopes . The latAltogether, these data indicate that the large clusters of the highly connected parts of the graph likely reflect the cross-reactivity of highly concentrated public anticarbohydrate antibodies and non-self-recognition.To focus on the specificities relevant to potential brain tumor classifiers, the peptide reactivities in the individual cliques were further tested for their capacity to separate the three diagnostic groups. The diagnoses were mapped onto the reactivity data for each 3-clique or the mean of all combinations of three peptides in the larger cliques. Sampling of the cliques larger than three vertices was done to compare the separation at the same dimensionality . A compo4 random samples of peptides of the same size as the tested clique. The selected 130 potentially diagnostic cliques (DClq) of reactivities were of sizes 3 to 7 . These represented 228 peptides. The predominant motifs extracted from these peptides using the MEME suit of algorithms are listed in The potential correlation of some IgM reactivities with the presence of tumors was further explored by focusing on the candidate peptides thus identified. To this end, the clustering criterion calculated on the reactivity data for each clique of size between 3 and 10 was compared to a bootstrapped 99th percentile of the clustering criterion on the reactivity data for 10The antigens significantly overrepresented in the GpClq cliques included 20/24 self-antigens and 8/29 non-self-antigens including the linker used in the microarray (GSGSGS). The latter was meant to be an inert flexible linker used as a separator between the protein sequences by the peptide manufacturer. Unexpectedly, it showed not only very high reactivity with human IgM but also involvement in cancer-induced IgM changes and is the most prominent motif in the set of DClq . Apart fThus, the diagnostically relevant reactivities included disproportionately more self-antigens. The viral epitopes found in the cliques were from EBV (EBNA and BZLF1) and HPV16 (E6 and E7), with one from human herpesvirus 8 LANA and one from HTLV1 Env. The most represented self-antigens were (in this order) Myc, G2/mitotic-specific cyclin-B1 (CCNB1), tyrosinase (TYRO), ErbB2, PSA, stromelysin (MMP11), and p53. These associations reached statistical significance for the following antigens and cliques: CCNB1 , Myc , TYRO , H4 , PSA , ErbB2 (23), SSX2 (9), CEA (9), and the viral epitopes from EBV , HPV , and HHV8/KSV (2) . The conInterestingly, the HTLV1 reactivity segregated from the widespread herpes and papilloma virus reactivities and with the self-antigens along the 6th component. This strengthened the hypothesis that some cross-reactivity with self may explain its central role while the virus is unknown to the population studied.Finding that reactivity graph clique sorting by size provides a suitable correlate for potential tumor-associated reactivities suggested a tool for filtering the IgM IgOme features to be used in a subsequent machine learning model.2–103 peptides) out of a collection of n × 103 peptides. This is necessary because of the astronomical number of possible combinations of features in the ultimate classifier of suitable size [High-throughput IgM binding data can be utilized as a source of biomarkers representing subsets of peptide reactivities (immunosignatures ) that coble size . The genble size ,35. A mable size  to derivble size . To prodp < 0.05). Thirteen of the 24 self-antigens were represented, and none showed a correlation with a particular PrCl. On the other hand, the viral antigen epitopes  were found mostly in PrCl 5 with increased reactivity in brain tumors, especially in metastases (The 51 peptides’ reactivities clustered in eight profile clusters (PrCl), five of which  showed a significant mean reduction in reactivity in the tumor groups compared to the control group, while only one (5) showed a significant increase in the GBM group . These characteristics combined with different accessibility of tissue antigens under different conditions may explain the number of reactivities found in non-tumor bearing patients and lost in those with tumors. This is also in line with previous findings of numerous IgM reactivities involved in tumor surveillance and tumor diagnosis. Disappearing IgM reactivities is an interesting recurring observation of the nature of the changes in the IgM repertoire in pathology ,27. It mThe IgM repertoire consists to a great extent of inherently polyspecific clones, and most of the bound structures are expected to be conformational epitopes. These two facts should be reflected in the reactivities to the 15-me epitopes of known TAA tested. While some of the observed reactivities may be actual autoreactivities to accessible epitopes involved in tumor surveillance, others may be simply cross-reactivities apparent only in vitro with little significance in vivo.Physiologically autoreactive antibodies could tag native tumor-associated antigens (TAA) simply as a result of overexpression or ectopic presence. Quantitative self-tolerance is comprehensible mostly in terms of physiologically maintained moderate to low titers of physiologically autoreactive antibodies . TherefoThere is a sufficient body of evidence for positive selection of the B cell repertoire on self-antigens ,44,45,46Rarely has IgM reactivity been used to extract diagnostic reactivity profiles. In mice, IgM reactivity to a set of autoantigens provided more consistent tumor-related patterns than IgG ,38,39. S102), as is its capacity to produce false classifiers by overfitting to the training set. What is somewhat unexpected is that the uncovered diverse sets of features would together yield an accuracy of exact classification in LOOCV with such small training sets. It is likely that many diverse classifiers can be found for the same task. A possible explanation would be that the IgM repertoire readily provides different efficient classifiers, which would converge on a common public repertoire profile as the number of training sets increases. In other words, the data suggest that a number of partially overlapping “repertoire phenotypes” exist among individuals. Some of these “repertoire phenotypes” may be temporary states of the repertoire that oscillate in time [Finding classifiers working well with the training set when using efficient feature selection algorithms is not so surprising. The space of subsets of more than 42 features out of 4524 is for all practical purposes infinite , brain metastases of prostate or lung cancers , and non-tumor-bearing patients  with herniated disc surgery, trauma, etc. The sera were aliquoted and stored at −20°C. Before staining, the sera were thawed; incubated for 30 min at 37 °C for dissolution of IgM complexes; diluted 1:100 PBS, pH 7.4, 0.05% Tween 20 with 0.1% BSA, further incubated for 30 min at 37°C, and filtered through 0.22 μm filters before use.® containing 4526 peptides produced by PEPperPRINTTM  were used. The peptides were synthesized in situ attached to the surface through their C-terminus and a common spacer GGGS and were included in the microarray layout in duplicate. The majority of the peptides represent a scan of the sequences of 24 tumor-associated antigens using a window of 15 amino acid residues shifted by two residues  on a rocker overnight at 4 °C. After 3 × 1 min washing, the chips were incubated with secondary antibodies at room temperature (RT), washed, rinsed with distilled water, and dried by spinning in a vertical position in empty 50 mL test tubes at 100× https://github.com/ansts/TAAIgM, last accessed 31 December 2022). The details of the analysis procedures are described elsewhere [The microarray images were acquired using a GenePix 4000 Microarray Scanner . The densitometry was done using GenePix R Pro v6.0 software. All further analysis was performed using publicly available packages of the R statistical environment for Windows (v3.4.1)  as well as in-house developed R scripts  better than the 95th percentile of the distribution of the criterion calculated from mappings on random samples of reactivities of the same dimensionality (the same number of 3 or 4 peptides) were filtered as candidates for the classifier.The selected features were further subjected to feature selection by recursive elimination. The criterion for selection was based on dichotomous supervised clustering of the patients in the different diagnostic groups (GBM vs. Control plus Metastatic or Control vs. tumor bearing). The goal was optimal clustering of the predetermined diagnostic groups estimated using a composed criterion based on 3 internal clustering indices: Baker–Hubert gamma , Dunn 5,51, and The significance of the approach and the selected feature sets were validated in a “leave-one-out” cross-validation (LOOVC) scheme encompassing the whole algorithm staring with the generation of the graph. Thus, the algorithm of a filter and a wrapper stage of the feature selection described above was applied successively to all sets of 20 patients excluding in turn each of them, and the capacity of the resulting feature set to predict correctly the excluded case was tested using a support vector machine model of the data projected to 2 dimensions using multidimensional scaling. The criteria for the efficiency of the classifier were F1 and Matthew’s clustering criterion calculated on the basis of the confusion matrix of prediction versus ground truth. Once the algorithm was validated, the ultimate model was constructed from features that recurred in the feature sets from the LOOCV. The threshold for the number of recurrences was determined empirically by scanning the different thresholds with the same clustering criterion.The proposed algorithm for the extraction of IgM repertoire-based diagnostic classifiers has the potential to provide a fast, non-invasive, and inexpensive tool for the analysis of malignant tumors. By using natural peptides from known tumor-associated antigens, it is possible to find interpretable reactivities pointing to known or unknown prognostic markers, which would give new opportunities in the diagnosis and treatment with possibly wider application in tumor immunology.


ELISA and IHC tests performed postmortem on the medial retropharyngeal lymph nodes (RPLN) or obex are considered diagnostic gold standards for prion detection. However, differences in CWD transmission, stage of infection, pathogenesis, and strain can limit performance. To overcome these uncertainties, we used Bayesian statistics to assess the accuracy of RT-QuIC, an increasingly used prion amplification assay, to diagnose CWD on tonsil (TLN), parotid (PLN) and submandibular lymph nodes (SMLN), and ELISA/IHC on RPLN of white-tailed deer (WTD) sampled from Minnesota. Dichotomous RT-QuIC and ELISA/IHC results from wild (n = 61) and captive (n = 46) WTD were analyzed with two-dependent-test, one-population models. RT-QuIC performed on TLN and SMLN of the wild WTD population had similar sensitivity (median range (MR): 92.2–95.1) to ELISA/IHC on RPLN (MR: 91.1–92.3). Slightly lower (4–7%) sensitivity estimates were obtained from farmed animal and PLN models. RT-QuIC specificity estimates were high (MR: 94.5–98.5%) and similar to ELISA/IHC estimates (MR: 95.7–97.6%) in all models. This study offers new insights on RT-QuIC and ELISA/IHC performance at the population level and under field conditions, an important step in CWD diagnosis and management.Chronic wasting disease (CWD) is a transmissible prion disease of the  Chronic Wasting Disease (CWD) is an incurable, progressive, and fatal prion disease affecting cervids worldwide. Since first reported in the 1960s in the U.S. , CWD hasThe accuracy of a diagnostic test is often estimated relative to a reference test, known as the gold standard. The “gold standard” assumes perfect accuracy in sensitivity, in which infected animals will always be detected by the test, and specificity, in which uninfected animals will never test positive. However, under natural conditions in which the infected population is a heterogeneous mix of animals at various stages of infection or disease progression and true disease status is unknown, disease status  misclassifications can occur, even when using the presumed gold standard approaches to disease detection ,30. CurrThe need for a faster, accurate, reliable, cost-effective assay, with the potential for application in antemortem diagnosis, has led to the development of techniques relying on prion amplification, such as the real-time quaking-induced conversion (RT-QuIC) . RT-QuICGiven the aforementioned challenges, knowledge of diagnostic test accuracy, as they are applied under conditions of natural transmission, is critically needed by researchers, regulatory agencies, and policy makers. A Bayesian latent class analysis (BLCA) is a useful alternative approach to the assessment of diagnostic test performance that can overcome the constraints related to reliance on a “gold standard” comparison ,47,48,49In this study, we aimed to evaluate the performance of RT-QuIC and ELISA/IHC for the detection of PrPSc in lymphoid tissues from white-tailed deer under field conditions. To do so, samples were collected from wild and captive white-tailed deer in Minnesota to a) quantify the concordance between RT-QuIC and the ELISA/IHC across different sample matrices, and b) estimate the sensitivity and specificity of RT-QuIC and ELISA/IHC using BLCA as compared to the traditional “gold standard” approach. Given its demonstrated capability for earlier prion detection in tissues of infected animals , we hypon = 61) white-tailed deer (WTD) in southeast Minnesota where CWD infection is persisting in approximately 1% of the WTD population (herein referred to as the “wild population”), and a depopulated CWD + herd of captive WTD (n = 46) in the same region (herein referred to as the “captive population”).RPLN, parotid lymph nodes (PLN), palatine tonsils (TLN), and submandibular lymph node (SMLN) tissue samples were obtained from culled, wild (n = 6) as PrPSc-positive than the standard approach. These included one PLN- and two TLN-positive animals from the wild, and two PLN- and one TLN-positive captive animals. When the RT-QuIC assay was performed on SMLN from the wild population, there were no inconclusive results to assess the different classification approaches.As an initial step in evaluating the dichotomous results for CWD diagnosis across tissues and tests, we created contingency tables comparing results from ELISA/IHC performed on RPLN, and those results from RT-QuIC performed on PLN and TLN in the captive population, and on PLN, TLN, and SMLN from the wild population. Results obtained from RT-QuIC were cross-tabulated considering two classification approaches: the standard, in which inconclusive results were computed as negatives, and the inclusive, in which inconclusive results were considered positives . The incWe examined the level of agreement between RT-QuIC by both classification approaches and the various tissue types and ELISA/IHC of the RPLN. To do this, we assessed the concordance among different diagnostic test combinations beyond chance using kappa statistics (κ). Agreement observed between RT-QuIC using different tissue matrices ranged between good and excellent (κ: 0.69–0.95), with the least agreement observed between RT-QuIC performed on PLN vs. TLN . The conFor demonstrative purposes, we first estimated the accuracy of RT-QuIC for PrPSc detection using the traditional “gold standard” approach. The performance of RT-QuIC in reference to ELISA/IHC as the gold standard tests of comparison showed a high sensitivity (Se) when using TLN and SMLN . RT-QuICTo avoid the “gold standard” assumption, we used a series of two-dependent-test, one-population latent class models to estimate the diagnostic Se and Sp of RT-QuIC (by both result classification approaches), ELISA and IHC, as well as population CWD prevalence. The statistical models combine results from two different diagnostic tests; ELISA or IHC as test one, and RT-QuIC for each tissue sampled respectively as test two, in each population of study independently (wild or captive), with prior information on each parameter to obtain the most probable field values. Prior information informs the most likely range of values for each parameter that are compatible with previously published studies and experts’ opinions while incorporating the knowledge uncertainty . Hence, informative, weakly informative, or non-informative priors were selected based on a lower to a higher level of uncertainty on previous knowledge and experiences.Latent class models yielded median Se estimates for ELISA/IHC of RPLN between 91 and 94.5% for the wild, and 88.4 and 93.1% for the captive population. Sensitivity of RT-QuIC resulted in higher (92.2–95.1%), similar (92.1%), or lower (88.2–88.3%) median estimates when tested on TLN, SMLN, and PLN, respectively, for the wild population. Lower Se-median estimates were observed when using RT-QuIC in TLN (88.5%) or PLN (73.8–74.3%) in the captive population. The inclusive classification approach for CWD results by RT-QuIC assay resulted in the highest Se : 76.6–99.8) when applied to TLN in the wild population model, with no differences observed in the estimates generated by standard vs. inclusive criteria in the rest of the latent class models using other tissues or the captive population . SpecifiWe structured our models assuming a conditional correlation between tests. This means that we assumed that if an infected animal tested positive  by one test , a similar result would likely be observed by the other test (RT-QuIC) , and a similar scenario will be observed for uninfected animals . This was primarily based on the fact that each test is based on the detection of the infectious prion in tissue . However, low conditional correlation estimates were observed between RT-QuIC and ELISA/IHC among infected and non-infected animals, as denoted by the 95% PPI including zero for all models .We conducted a sensitivity analysis to determine how much our model structure and prior parameter distributions influenced model results. We did this by running a new set of models that included a two-population model and where uninformative priors were reassigned to individual parameters. Specifically, beta distributions around prior median estimates of Se, Sp, and prevalence were replaced with uninformative uniform distributions . Sensitivity analyses indicated no effect on the median posteriors of the parameters estimated (variations < 10%) when fitting a two-dependent test, two-population model, however, the DIC was higher (>10 points) in the latter model , which iWith the development and application of new diagnostic tests for CWD and other prion diseases, there is a critical need to understand the efficacy of their performance in naturally infected populations, where true disease status is unknown. With a recognition of imperfect testing for disease classification, the current study applied Bayesian statistics to determine the accuracy and best use of two diagnostic assays for CWD in free-ranging and captive WTD. Through these analyses, we demonstrate high Se and Sp of RT-QuIC, particularly when used on TLN, and provide the first set of performance estimates for RT-QuIC as well as ELISA/IHC in both wild and captive settings. We employed this approach in association with the frequently used assessments of test performance, including analysis of test agreement and in reference to an assumed “gold standard” of comparison. While the accuracy of RT-QuIC in reference to the ELISA/IHC as the “gold standard” showed similar patterns of performance to those observed with the BLCA, they were characterized by higher uncertainty and limited validity under natural conditions . Indeed,Our estimates of ELISA/IHC test performance are the first quantitative estimates of the accuracy of these tests for PrPSc detection in a natural setting. Although the limitations of these tests for diagnosis in cervid populations have been described ,11,12,13n = 5, RT-QuIC exhibited the highest level of Se when performed on TLN and applying an inclusive criterion of classification. Currently, there is a lack of standardization among the research and diagnostic community on the assignment of the dichotomous classification of CWD disease status (positive vs. non-detected) from the amyloid formation process of RT-QuIC. Up to now, a variety of statistical approaches have been employed for disease status classification, such as Mann-Whitney ,53, Wilcn = 5) biopsies of TLN in CWD-infected WTD [Diagnosis reached with RT-QuIC using TLN and SMLN had higher than or similar Se and Sp for CWD diagnosis as ELISA/IHC using RPLN. The high sensitivity observed with TLN samples replicated results observed in previous studies of IHC performed on limited  with oropharyngeal prion entry ,60, or tConsistently higher posterior RT-QuIC Se estimates of the wild vs. captive population indicate different levels of detection by the same diagnostic test according to the population under study. This finding suggests that these two populations may vary in their proportions of animals at certain stages of the disease, PrPSc strains, or predominant routes of prion transmission. Certainly, the route of transmission and time post-infection can influence the prion load in the lymphoid tissues under study, and that can impact the accuracy of tests for identifying infected animals ,34. In a1, Se2, Sp1, Sp2, rhoD, rhoDc, Prev) was preferred over an eight-parameter one . The low correlation observed between tests results, although not initially expected, could be due to different levels of PrPSc concentration across the lymphoid tissues used with each test type, impacting disease detection [To estimate the sensitivity and specificity of RT-QuIC and ELISA/IHC, we fitted models using one population to ease interpretation and because, based on DIC statistics, a seven-parameter model  for the Se and Sp estimates for ELISA/IHC because of the limited studies of performance available, of which the majority were applied under laboratory conditions or with limited sample size ,31,32,64Priors established for the population prevalence of the wild WTD models were higher than those reported for the southeast Minnesota WTD wild population, and more similar to those observed in depopulated captive populations in the state . Indeed,Guidelines from the STARD-BLCM were followed to describe the materials and methods in our study .Samples of lymphoid tissue were collected in 2019 (wild population) and 2021 (captive population) from two distinct WTD populations in Minnesota (MN), with a cross-sectional study design.Lymphoid tissues were sampled from 46 culled captive asymptomatic animals originating from a CWD outbreak farm previously detected in southeastern MN. The farm was confirmed CWD-positive by the Minnesota Board of Animal Health (BAH) and United States Department of Agriculture (USDA); tissues were collected at the time of depopulation for the control of CWD . FurtherOfficial authorities (MNBAH and MNDNR) collected RPLN for CWD detection in both populations of WTD. Bilateral RPLN collected from captive WTD were diagnosed by IHC at the National Veterinary Services Laboratory . Bilateral RPLN of wild WTD were submitted for diagnosis at the Colorado State University Veterinary Diagnostic Laboratory  for screening by ELISA, using the Bio-Rad TeSeE Short Assay Protocol (SAP) Combo Kit . For each animal, a homogenate was produced using three subsamples from each RPLN and tested as a pooled sample of both RPLN subsamples. ELISA-positive samples were confirmed by IHC, as described . Samplesw:v) suspension was made by adding 100 mg of tissue to 900 µL of phosphate-buffered saline (PBS). Homogenized samples were diluted further to 10−3 in dilution buffer ), and 2 µL were added to 98 µL of RT-QuIC master mix  at 700 rpm, double orbital for 57 s, then rested for 83 s, repeated 21 times, then the fluorescence was recorded. The temperature was set at 42 °C and the cycles were repeated over 46 h.Bilateral lymphoid tissue from the palatine tonsils (TLN), parotid (PLN), and submandibular lymph nodes (SMLN) (the latter only for the wild population) were sampled, pooled into a single homogenate, and screened by RT-QuIC by the Minnesota Center for Prion Research and Outreach (MNPRO) laboratories using a blinded approach. RPLN were not available from either population for RT-QuIC analysis as they were used for the regulatory testing (IHC and/or ELISA). The sample collection and RT-QuIC methodology have been described previously . Brieflyin house ) into eap-value ≤ 0.05) higher intensity than the negative control. Analyses were performed using GraphPad Prism software , as described previously [RT-QuIC results were dichotomized using two criteria, standard and inclusive. The standard approach classified as positive those samples in which at least one of the sample quadruplicates tested showed an amyloid formation as relative fluorescent units in a sample over time with significantly , and ELISA/IHC from RPLN were estimated using kappa statistics  applied Analytical sensitivity and specificity of RT-QuIC in different lymphoid tissues were initially assessed using ELISA/IHC in RPLN as a gold standard diagnostic reference and calculated using Epitools Epidemiological Calculators .Two-dependent test, one-population Bayesian latent class models were applied in the absence of a gold standard to estimate diagnostic Se, and Sp of RT-QuIC (using standard and inclusive classification approaches), as test one, and ELISA in the wild or IHC in the captive WTD population, as test two in each model, respectively ,73. The The Se and Sp for the ELISA/IHC were informed by priors that followed the beta-distribution, built based on previous literature ,31,32. TModels were fitted and run using OpenBUGS 3.2.2  via the 2, Sp2, Prev1, Prev2). We had one exception to the latter approach where the model assessing the performance of RT-QuIC in TLN, where we substituted the uninformative uniform priors with weakly informative  beta priors informed by the limited number of studies from the literature  between the initial models and (a) two-dependent test, two-populations models with similar priors, and (b) two-dependent test, one-population model where non-informative priors  were substituted sequentially for the beta priors of each parameter under study (i.e., Seterature  [43,58,7To conclude, our robust results, supported by model checking and sensitivity analyses, revealed high diagnostic Se and Sp of RT-QuIC for PrPSc detection in TLN and SMLN under field conditions. We also show that RT-QuIC has improved performance over ELISA/IHC when the approach to dichotomous result classification is relaxed, an approach that can enhance Se without compromising Sp. Among the tissue types tested by RT-QuIC, the use of TLN and SMLN perform equivalently to ELISA/IHC of RPLN, with TLN demonstrating the highest Se of detection. Based on these results, we recommend avoiding the use of PLN for CWD diagnosis in WTD due to lower Se of detection. Our results build on the understanding of the performance of RT-QuIC and ELISA/IHC at the population level and under natural conditions, which represents an important step forward with respect to diagnostic tests for the management of CWD.