International Journal of Cancer (IJC), we provide an overview of the issues pertinent to misidentified cell lines and discuss available solutions. We also summarize the lessons learned, revealing that at least 5% of the human cell lines used in manuscripts considered for peer review are misidentified. About 4% of the considered manuscripts are rejected for severe cell line problems, and most are subsequently published in other journals. In order to diminish such malpractice and its consequences for the scientific record, we postulate that strict multi‐layered quality control is essential. Besides journals and publishers, we encourage scientists, research institutions, and funders to take action on the matter and revise their respective policies. Hence, we provide concrete recommendations on introducing regular authentication schemes and staff training, and discuss future steps for enhancing good cell culture practices.Immortalized or continuous cell lines are invaluable tools in basic and preclinical research. However, the widespread use of misidentified cell lines is a serious threat to scientific reproducibility. Based on the experiences of mandatory cell line authentication at the  This commentary discusses multi‐layered quality control of immortalized cell lines as an essential prerequisite for reliability of the scientific record and good cell culture practices. Philippe Gorphe identified 1,036 articles published between 1954 and January 2018 specifically referring to the wrong laryngeal origin of HEp‐2 cells. He also showed that the number of manuscripts using the HEp‐2 cell line as a laryngeal carcinoma has increased over the past three decades , cell line cross‐contamination decreased from 15 to 6%. However, among cell lines obtained from secondary sources , the cross‐contamination prevalence remained high at 14–18% over the 25‐year timespan, indicating that one in six of the secondarily sourced cell lines, shared between laboratories, is misidentified , including 848 leukemia–lymphoma cell lines received between 1990 and 2014 from 290 laboratories in 23 countries, showed a difference in the prevalence of cell line cross‐contamination depending on the cell line source. Among cell lines obtained from primary sources  raised alarm bells when they reported a misidentified cell line rate of 25% after profiling 380 cell lines obtained from 113 independent sources , the corresponding purchase orders or invoices are accepted for authentication.Of continuous human cell lines obtained within the last 3 years from a commercial source, which guarantees cell line authenticity through in‐house quality control measures , and should show that the cell line is unique and not cross‐contaminated or misidentified.The •IJC also accepts single nucleotide polymorphism (SNP) based cell line authentication reports from service providers with certified quality control, but only for cell lines of which an SNP‐based reference profile is publicly available.The •Authors of studies describing the establishment of new human cell lines are strongly encouraged to include the summarized STR results in the manuscript for future reference.•○e.g., HeLa (RRID:CVCL_0030)).All cell lines used must be listed using the official cell line name and its Research Resource Identifier (RRID) as available in the ExPASy Cellosaurus database  profiling within the last 3 years.○A statement confirming that all experiments were performed with mycoplasma‐free cells.Since May 2019, the following information must be included in the Materials and Methods section:aAdapted from https://onlinelibrary.wiley.com/page/journal/10970215/homepage/ForAuthors.html#AUCEL.bUntil May 2019 cell line authentication documents not older than 4 years were accepted.In this 3‐year period, 747 manuscripts containing original data on 4,138 human cell lines were considered for peer review. The majority of these submissions (∼65%) were not accompanied by valid cell line authentication documents and had to be unsubmitted to ask for correct documents, indicating that regular authentication of cell lines is not common practice. Eventually, valid authentication documents were obtained from 3,091 (74.7%) cell lines. By verifying the available cell line authentication documentation and cross‐checking the Cellosaurus database, 216 (5.2%) misidentified cell lines were identified, including 186 misidentified cell lines that were completely overgrown and replaced by a contaminator cell line, 16 mixtures of two cell lines, and 14 reported nonintentional hybrid cell lines Fig . The moset al, The highest percentage of misidentified cell lines was observed among gastric cancer (25.4%), followed by liver cancer 16%) cell lines Fig . Hence, 6% cell le.g., manipulated electropherograms or fake invoices).Based on the available information, no cell line‐related problems were observed in 77.1% of the considered manuscripts Fig . HoweverBox 2•No cell line problems: Based on the available cell line authentication documentation and by cross‐checking the Cellosaurus database, no cell line issues were observed.•Minor cell line problems: These manuscripts only needed minor textual clarifications. For instance, the authors used a cell line that is known to be misidentified by another cell line from the same tumor type, as recorded in the Cellosaurus database, and the authors were asked to add this information to their manuscript . Or the authors were asked to clarify that two of the used cell lines originate from the same donor .•Moderate cell line problems: One cell line had to be removed from the manuscript because it was misidentified by a cell line from a different tumor type, the authentication documents revealed that the sample contained a mixture of two cell lines, or the cell line is recorded in Cellosaurus as a reported nonintentional hybrid cell line. Moderate cell line issues also include manuscripts containing misidentified cell lines that did not need to be removed because they are known to be misidentified by a cell line from the same tumor type as recorded in Cellosaurus, but the misidentified cell line had a large impact on the significance of the manuscript . Moderate cell line issues also include manuscripts in which a cell line needed to be renamed because the authentication documents showed that the cell line was misidentified by another cell line from the same tumor type, but the cell line was not among the known misidentified cell lines recorded in Cellosaurus.•Severe cell line problems: At least two cell lines had to be removed from the manuscript because they were misidentified by a cell line from a different tumor type, the samples contained a mixture of two cell lines, and/or reported nonintentional hybrid cell lines were used. In case the misidentified cell line(s) affected a large amount of the presented data and/or the cell line authentication documentation was clearly falsified , then it was decided to reject the manuscript for severe cell line issues.The cell line‐related problems observed in the manuscripts that were considered for peer review and included original human cell line data were divided into the following categories:IJC with severe cell line‐related problems are subsequently published elsewhere, we followed up 63 manuscripts. These included 39 manuscripts from the analysis above that had been considered for peer review but were unsubmitted or rejected for serious cell line‐related problems between July 2018 and December 2020. The remaining 24 manuscripts that we followed up from this time period were not considered for peer review, but it was noticed that a large amount of the data were based on (known) misidentified cell lines. In all cases, the authors were clearly informed about the cell line problems observed in their manuscript in the unsubmission or rejection letter. Afterwards, we observed that 50 (79.4%) of these manuscripts were published in other journals, of which 45 (71.4%) manuscripts still included the data of the misidentified cell lines. Of these 45, 32 (50.8%) manuscripts included data of known misidentified cell lines with no known authentic stock, which means that the existing cell line problems could not have been solved in the meanwhile. Remarkably, in 5 (7.9%) cases, the data of known misidentified cell lines with no known authentic stock was renamed into another cell line. Additionally, one such manuscript was even resubmitted to the IJC. As the intentional publishing of misidentified cell line data represents scientific misconduct, the policy of the IJC is that new submissions of authors rendered guilty in this regard will not be considered. Because of data protection rules, the IJC cannot share personal data of authors with other journals. However, in order to correct the records, other journals are invited to contact the IJC to obtain general instructions on how they can identify published manuscripts that are based on known misidentified cell lines.In order to explore whether manuscripts rejected at the Our experiences show that the misidentified cell line problem is very persistent and that strict editorial quality control procedures are essential to prevent publishing data based on misidentified cell lines. In order to avoid the publication of studies including misidentified cell line data and to correct the contaminated published records, journals and publishers should play a more active role. Hence, specific recommendations and perspectives for journals and publishers are discussed below and summarized in Fig et al, Many commonly used cell line names are not unique Bairoch, . Hence, Moreover, about 60% of the misidentified cell lines identified in our evaluation are (now) known misidentified cell lines and are recorded in the Cellosaurus database. Therefore, journals can easily prevent publishing data based on these problematic cell lines by verifying all cell lines used in a manuscript in the Cellosaurus database even before starting the peer review process.Around 40% of the misidentified cell lines identified in our evaluation are not listed as known misidentified cell lines in the Cellosaurus database. This portion of misidentified cell lines can thus only be uncovered by implementing a strict mandatory cell line authentication policy. Verification of the authentication data by a qualified person is crucial because misidentified cell lines are often missed by authors due to misinterpretation of the STR results. We realize that verification of all cell line authentication documents will not be feasible for every journal. Nevertheless, if journals implement mandatory cell line authentication in combination with occasional verification of the documents, regular authentication of cell lines will become common practice and misidentified cell lines will be identified and eliminated earlier.Even a strict cell line policy cannot completely prevent publishing manuscripts based on misidentified cell lines, and we encountered several cases where a correction of already published manuscripts with cell line problems became necessary.et al, Exemplarily, we obtained a unique STR profile of the presumed lung cancer cell line SPC‐A1, of which no reference STR profile was publicly available and the obtained STR profile did not give a match in the available STR databases. Two years later, we reassessed the STR profile in the updated Cellosaurus CLASTR tool, which revealed that the used SPC‐A1 cell line was actually the hepatocellular carcinoma MHCC97‐H cell line , sometimes even accompanied by misleading cell line authentication reports. At the IJC, we accept purchase orders or invoices of authors who purchased their cell lines not longer than 3 years ago from commercial sources guaranteeing cell line authenticity through in‐house quality control measures, assuming that these sources would not distribute misidentified cell lines. Although our procedure has been adapted and invoices of such doubtful sources are now only accepted together with valid certificates of authentication, in the past manuscripts using these misidentified cell lines have been published and we are now in the process of correcting these records. However, publishing errata is a time‐consuming process, and there are hundreds of thousands of manuscripts published in other journals, based on known misidentified cell lines with no known authentic stock, that need to be corrected. Therefore, we support the idea of Horbach & Halffman  as Supplementary Material.Nowadays, many biomedical journals, including the IJC also receives studies that use cell lines of nonhuman origin, among which mouse cell lines are the most common. Although the species of origin and inter‐species cross‐contamination can easily be detected using a multiplex PCR‐based assay , and whenever a phenotypic change—particularly in growth behavior—is observed. In general, scientists are advised to acquire continuous cell lines directly from certified cell banks that guarantee cell line authenticity through internal STR analyses . If a cell line is obtained from a secondary source , the cell line should be quarantined antibiotic‐free until its identity has been confirmed by STR profiling, and the overall sterility and absence of mycoplasma have been verified.Cell lines in long‐term use should be authenticated at the beginning and the end of a project, especially after selection procedures . For future reference, summarized STR results should be included in the manuscript as an essential part of the initial description so that authentication data can be included in the Cellosaurus database and associated with the RRID.The establishment of new cell line models is an important but difficult task, as cross‐contamination at the source is one of the most common causes for the development of misidentified cell lines (MacLeod https://iclac.org/education/). For further and detailed instructions on good cell culture practice, we refer to the “Guidance document on good in vitro method practices (GIVIMP)” by the Organization for Economic Co‐operation and Development , the research institutions themselves, and finally, the publication bodies. In this respect, funding agencies can play a very important role if they require proof of authentication for the planned use of tumor models to be submitted at the time of application, as already practiced by the German Wilhelm Sander Foundation, the US National Institutes of Health, or the British Cancer Research UK.IJC, we notice that authentication of cell lines is a more common practice among researchers who are affiliated to research institutions with an own dedicated cell line authentication facility. This type of authentication infrastructure has long been established at large companies such as AMGEN, Merck, or Roche Holding, but is also increasingly provided at large research institutions such as the German DKFZ or MD Anderson Cancer Center in the USA. Internal STR profiling facilities allow easy access to rapid authentication testing and ensure the presence of in‐house expertise for the interpretation of challenging samples, such as hybrid cell lines or mixtures. Hence, research institutions should consider offering a cell line authentication service in their own core facility. In addition, biomedical research institutions should develop guidelines for cell line use, as implemented by the MD Anderson Cancer Center requiring all researchers to authenticate their cell lines once a year. For a draft, we refer the reader to Schweppe and Korch, who suggested a detailed cell line and tissue authentication policy for biomedical research institutions (Schweppe & Korch, At the “This analysis could become a prerequisite for publication, so that the problem of cell line cross‐contamination can be reduced to a minimum in the future” (Masters et al, IJC, still at least 5% of the human cell lines used in manuscripts considered for peer review are misidentified and about 4% of the considered manuscripts are rejected for severe cell line problems. The fact that the majority of the manuscripts unsubmitted or rejected for severe cell line problems have since been published in other journals, illustrates the irresponsibility of some authors toward the misidentified cell line problem. Hence, we call on scientists, research institutions, journals, and publishers to take action by implementing a mandatory cell line authentication policy and by developing a strategy to correct the contaminated literature. A collaborative approach that includes proactive funding agencies, rapid access to low‐cost authentication services, and widely requested proof of authentication from journals should significantly reduce the chronic dilemma of using misidentified cell lines in the future.Twenty years ago, when John Masters and colleagues introduced STR profiling as the international reference method for human cell line authentication, they were very optimistic and stated: Nicole Y Souren: Conceptualization; data curation; formal analysis; investigation; visualization; writing – original draft; writing – review & editing. Norbert E Fusenig: Conceptualization; supervision; writing – review & editing. Stefanie Heck: Conceptualization; data curation; investigation; writing – review & editing. Wilhelm G Dirks: Expert advice; visualization; writing – review & editing. Amanda Capes‐Davis: Expert advice; writing – review & editing. Franca Bianchini: Supervision; project administration; writing – review & editing. Christoph Plass: Conceptualization; supervision; project administration; writing – review & editing.The authors declare that they have no conflict of interest.


The authors declare that there is no conflict of interest.Dear Editor,A decrease in sperm quality that is reflected in alteration of all classical sperm parameters is now observed in developed countries, and has been attributed to exposure to endocrine disruptor chemicals (EDCs). EDCs are also present in maternal body fluids and in embryos , is the unique effector for methylation. Methylation of target molecules results in the formation of SAH (S‐adenosyl homocysteine) and release of homocysteine Figure . Hcy is The systemic UMFA concentration is directly influenced by folic acid intake; FA supplementation with >1000 µG during pregnancy may lower cognitive development in children (Valera‐Gran et al.,


In Germany, geriatricians deliver acute geriatric care during an acute hospital stay and subacute rehabilitation after transfer to a rehabilitation clinic. However, the proportion of patients who receive acute geriatric care (AGC) or are transferred to subacute rehabilitation (TSR) differs considerably between hospitals. The aim of this study was to analyse the association between the two geriatric treatment systems and care home admission or mortality in patients following hip fracture.Health insurance claims data were used to identify the rate of AGC per hospital and the rate of TSR per hospital following hip fracture surgery in patients aged ≥ 80 years. Outcomes were cumulative admission to a care home and cumulative mortality within 6 months after hospital admission.Data from 23,046 hip fracture patients from 561 hospitals were analysed. The rate of AGC was not associated with care home admission. However, compared to high rates of AGC medium rates or no AGC were associated with increased death rates by 12% or 20%, respectively. Treatment in hospitals with low rates of TSR was associated with a 8% higher risk of care home admission and a 10% increased risk of death compared to treatment in hospitals with high rates of TSR.Our study suggests potential effects of geriatric treatment: reduction of mortality in hospitals with high rates of AGC or reduction of care home admission and mortality in hospitals with high rates of TSR. Hip fracture is associated with increased mortality, loss of independence and care home admission , 2. MostIn Germany, geriatricians deliver both ‘acute geriatric care’ (AGC) during the acute hospital stay and subacute geriatric rehabilitation after ‘transfer to a subacute rehabilitation’ clinic (TSR). AGC in patients with hip fracture is delivered in cooperation with orthopedic surgeons as orthogeriatric comanagement. It begins soon after surgery and lasts at least 14 days due to the current DRG definition. An accelerated discharge would lead to a drastic loss in reimbursement. This is acknowledged as an artefact but a widely accepted practice. The geriatric treatment is provided by a multidisciplinary geriatric team led by a geriatrician and made up of physiotherapists, occupational therapists, specifically trained nurses, social workers, and additional disciplines if needed. It includes a standardized comprehensive geriatric assessment, regular interdisciplinary team meetings, development of a rehabilitation plan with setting of functional goals and a medical focus on geriatric syndromes. Early mobilisation is a key component of the treatment procedure. In patients with hip fracture, AGC can be delivered on an orthopedic or a geriatric unit. In Germany, the two most common models are either shared responsibility on an orthogeriatric unit (integrated model) or a geriatric liaison service on the orthopedic unit with a rapid transfer to an acute geriatric unit .An alternative model or sometimes additional treatment is delivered in subacute geriatric rehabilitation units. Hip fracture patients are usually transferred to a subacute geriatric rehabilitation clinic after an acute hospital stay of 7 to 14 days. These patients require a pre-defined functional status, which excludes patients with a very low functional status, e.g. a Barthel Index < 40. The subacute rehabilitation lasts usually 3 weeks. Sometimes it can be extended by 1 or 2 weeks. The rehabilitation is based on a comprehensive geriatric assessment and is also provided by a multidisciplinary geriatric team. Regular interdisciplinary team meetings, development of a rehabilitation plan with setting of functional goals and a medical focus on geriatric syndromes are basic parts of the rehabilitation process.and TSR. But if a hospital offered AGC or TSR did not naturally mean that all hip fracture patients actually received one or even both types of geriatric treatment. Potential reasons were, that the patient’s functional status may not have qualified for the specific geriatric treatment, the  infrastructure of the hospital may not have been sufficient to offer AGC to all patients, subacute geriatric rehabilitation clinics may not have been available in the nearer region and transferring hip fracture patients to subacute geriatric rehabilitation clinics may not have been a tradition in some hospitals so far. Therefore, the rates of AGC or of TSR per hospital were clearly below 100% and differed from hospital to hospital considerably.In the past, most hospitals offered either one or the other type of geriatric treatment according to the federal state in which they were located. Since 2012 this historical situation has changed considerably. Many hospitals from states offering ‘subacute geriatric rehabilitation’ established AGC whilst the states offering AGC were required by law to develop subacute geriatric rehabilitation services. During this transition period hip fracture patients were treated at their acute stay in hospitals which delivered a) exclusively AGC or b) exclusively TSR or c) both AGC A comparison of the different geriatric treatment systems on health-related outcomes like care home admission or mortality has rarely been undertaken. In patients with hip fracture, rates of care home admission or mortality may reflect (achieved) functional status or hospital’s quality of care and are regarded as outcomes of high relevance.The heterogeneous situation of geriatric treatment in Germany can now be regarded as a ‘natural experiment’. We used this ‘natural experiment’ and performed a study based on health claims data to analyse the relationship between the two geriatric treatment systems on care home admission and mortality in patients aged ≥ 80 years with hip fracture.The basic dataset consisted of 87,022 patients aged ≥ 65 years admitted to hospitals with a femoral fracture between 01.01.2015 and 30.06.2016 in Germany and insured by the “Allgemeine Ortskrankenkasse” (AOK) insurance company. AOK is Germany’s largest health insurance company and covers nearly one-third of Germany’s 82.5 million population. Patient-related health insurance claims data were provided by the scientific institute of the AOK  in de facto anonymized manner.n < 80 per year) were excluded. Furthermore, patients who were residents of a care home before hospital admission were excluded.Hip fractures were identified using hospital admission diagnostic codes S72.0 and S72.1 (ICD-10)  can be identified in insurance health claims data by the German procedure classification code ‘OPS8-550’. It represents a complex treatment of early rehabilitation lasting at least 14 days  misclassification of exposure and b) selection bias of transferred (and not transferred) patients Fig. .After the acute hospital stay patients can be transferred to a subacute rehabilitation clinic (TSR). This information is recorded in routine hospital datasets permitting calculation of the rate of TSR per hospital. Subacute geriatric rehabilitation needs an upfront approval by the health insurance, is delivered over a time period of 3 weeks and is sometimes extended by 1 or 2 weeks (see Table Independent variables were 1.) the rate of AGC (OPS 8–550) per hospital and 2.) the rate of TSR per hospital; calculated as the number of hip fracture patients with an AGC (or a TSR) in each hospital divided by the total number of hip fracture patients in the same hospital. Each variable was categorized in three groups: none (0%), medium (> 0 to ≤ 48.8%) and high (> 48.8%) rates of AGC, and low (< 18.6%), medium (18.6 to 43.6%) and high (> 43.6%) rates of TSR. For AGC the chosen categories followed a dichotomization (medium and high), for TSR a tercile split.The OPS8-550 procedure code and information about transfers to a subacute rehabilitation clinic were used to characterize the degree to which hospitals provided AGC or TSR for their patients. Whilst, at first sight, it might seem straightforward to base analysis on individual-level data based directly on the presence or absence of OPS8-550 codes or the information about a transfer to a subacute rehabilitation clinic in individuals’ claims records, this approach is misleading and therefore not appropriate. Reasons are that a) individual allocation on the basis of OPS8-550 coding would introduce immortal time bias (survivorship bias)  since toThe outcome was cumulative admission to a care home within 6 months following hospital admission due to a hip fracture. Since AGC (qua surrogate of orthogeriatric co-management) is associated with reduced mortality , 9 diffeSozialgesetzbuch’). In order to claim for long-term care benefit, people must need daily a minimum of 90 min of assistance with basic activities of daily living such as washing, eating, or dressing and of instrumental activities of daily living such as cleaning or shopping. Verification and assessment of care need is performed by the medical service of the health insurance funds. Depending on the extent of care required recipients are categorised into 3 degrees of care need. This classification of care need can be used as a surrogate marker of disability [Age in years at the index fracture and sex were documented in the claims database. An assessment of the degree of care need is a requirement for those identified as “frail care recipients “ under German Social Security Code XI . All models were adjusted for age, sex, need for care on the day before fracture, the number of hip fracture patients/hospital/year, days from hospital admission to surgery, and medication-based comorbidity score. In all models, the continuous variables  were standardized to have mean zero and unit variance.Multilevel binomial regression models with hospital-specific random effects were used to estimate incidence ratios with 95% confidence intervals  and R version 4.0.2 .The analyses included 23,046 patients aged ≥ 80 years from 561 hospitals from 16 federal states which make up the Federal Republic of Germany. When analysing the influence of different types of geriatric treatment on outcomes on a systemic level, the three most prevalent combinations of geriatric health care after hip fracture were: rates of AGC high with TSR low; AGC medium with TSR high; and AGC medium with TSR medium  Table .Treatment in hospitals with low rates of TSR was associated with a 8% higher risk of care home admission compared to treatment in hospitals with high rates of TSR (IR (95% CI) 1.08 (1.00–1.16)) after full adjustment for covariates Table . HospitaTable We analysed the association between the availability of two geriatric treatment systems used in routine medical care in Germany on care home admission and mortality in patients aged ≥ 80 years with hip fracture. AGC was not associated with care home admission but was associated with a reduction of death rate in a dose–response manner. Furthermore, low rates of TSR were associated with higher care home admission and mortality.Notably, our results do not describe the risks or benefits for any single individual who receives AGC or is transferred to subacute rehabilitation. Since both geriatric treatment systems require a pre-defined functional status an approach which analyses the effects of AGC or subacute geriatric rehabilitation at an individual level would introduce strong selection bias. Instead, we used a systemic approach and analysed the potential benefits of the two different geriatric treatment systems according to their availability in the hospitals in which patients with hip fracture were treated. Therefore, the degree of individual benefit of receiving one or the other geriatric treatment is probably even higher than our reported estimates.Furthermore, mortality during the acute hospital stay may affect the rate of care home admission since both are associated with comorbidity and functional disability and perhaps more importantly to be discharged to a care home the patient has to be alive , 15. ThiOver recent years there has been growing evidence from randomized , 9 and lA reduction of care home admission by subacute geriatric rehabilitation is also supported by another study from Germany which used historical data. It compared federal states with different geriatric treatment systems and observed the lowest rate of care home admission in the federal state with the highest rate of subacute geriatric rehabilitation .Strengths of our study are the large number of included hospitals and patients, and the chosen methodical approach to analyse the scientific question on a systemic level instead of an individual level. In our opinion, this approach is robust and conservative and reduces the risk of selection bias considerably.Care home admission is clearly influenced by personal and contextual factors. Since we used health claims data we were not able to adjust for potential confounders like cognition or social support. In addition, the analysis may have been biased by the presence of competing risks not accounted for. On the one hand, it is the nature of our observational study to analyse associations which may be influenced by the specific structure of the German health care system. Naturally, the generalisability of the study results to other countries may therefore be limited. On the other hand, AGC and subacute geriatric rehabilitation represent two basic principles of geriatric care which are present in many countries of the industrialised world.In summary, we used a ‘natural experiment’ to analyse the effect of two geriatric treatment systems on care home admission and mortality in patients aged ≥ 80 years following hip fracture. Our study demonstrated potential effects of geriatric care: lower mortality rates in hospitals with high rates of acute geriatric care and lower rates of care home admission and mortality in hospitals with high transfer rates to subacute rehabilitation.


Despite the wide adoption of insertable cardiac monitors (ICMs), high false‐positive rates, suboptimal signal quality, limited ability to detect atrial flutter, and lack of remote programming remain challenging. The LUX‐Dx PERFORM study was designed to evaluate novel technologies engineered to address these issues. Here, we present preliminary results from the trial focusing on the safety of ICM insertion, remote monitoring rates, and the feasibility of remote programming. LUX‐Dx PERFORM is a multicenter, prospective, single‐arm, post‐market, observational study with planned enrollment of up to 827 patients from 35 sites in North America. A preliminary cohort consisting of the first 369 patients who were enrolled between March and October 2021 was selected for analysis. Three hundred sixty‐three (363) patients had ICM insertions across inpatient and outpatient settings. The mean time followed was 103.4 ± 61.8 days per patient. The total infection rate was 0.8% (3/363). Interim results show high levels of remote monitoring with a median 94% of days with data transmission (interquartile range: 82–99). Thirteen (13) in‐clinic and 24 remote programming sessions were reported in 34 subjects. Reprogramming examples are presented to highlight signal quality, the ability to detect atrial flutter, and the positive impact of remote programming on patient management. Interim results from LUX‐Dx PERFORM study demonstrate the safety of insertion, high data transmission rates, the ability to detect atrial flutter, and the feasibility of remote programming to optimize arrhythmia detection and improve clinical workflow. Future results from LUX‐Dx PERFORM will further characterize improvements in signal quality and arrhythmia detection. Their utility in the diagnosis of arrhythmias in clinical contexts such as syncope, cryptogenic stroke, and rhythm management before and after ablation has been well established. They have been shown to determine causation and impact management in significant numbers of patients.We have recently published the in silico experience with a new ICM, the LUX‐Dx.The LUX‐Dx PERFORM trial is the in vivo follow‐up to our previous study and is designed to evaluate the safety of the ICM insertion, the performance of arrhythmia detection algorithms, and the feasibility of remote programming. In this report, we evaluate the safety of ICM insertion, remote monitoring rates, and the feasibility of remote programming in the first cohort of patients enrolled in the LUX‐Dx PERFORM trial.22.1The LUX‐Dx PERFORM Study is multicenter, prospective, single‐arm, post‐market, observational study . The trial is being conducted at up to 35 sites in the US and will enroll approximately 600 to 827 subjects. A cohort consisting of the first 369 enrolled patients was selected for interim reporting. The investigation conforms to the principles outlined in the Declaration of Helsinki, Institutional Review Board approval has been obtained at each center, and all patients provided written informed consent.2.2Subjects are selected from the investigators' patient population per labeled indication for LUX‐Dx ICM insertion. The LUX‐Dx ICM System is indicated to monitor and record Subcutaneous ECGs for the clinical evaluation and diagnosis of cardiac arrhythmias in patients who have: 1) a known heart condition, (2) are at risk of developing an abnormal heart rhythm, or (3) have symptoms that may suggest a cardiac arrhythmia . All patients who meet all the inclusion criteria and none of the exclusion criteria may be given consideration for participation (Supporting Information: Table  a known 2.3The LUX‐Dx ICM (Boston Scientific Corporation) uses a data management system, LATITUDE Clarity™, which allows clinicians to remotely monitor and program the ICM. To transfer the ICM device data to LATITUDE Clarity, the system utilizes the myLUX™ Patient mobile app and the LUX‐Dx Clinic Assistant mobile app on dedicated smartphones. This allows clinicians to access the device data for analysis to support their evaluation of the clinical status and to adjust the diagnostic programmable features as needed  and communicate with the subject via telephone calls/virtual health visit or in‐office visit at two primary time points (6 and 12 months after insertion) for documentation of relevant adverse events. For this study, these follow‐up visits do not require subjects' physical presence at the study site. A cohort of approximately 600 subjects will wear an extended Holter monitor (HM) for approximately 14 days. The HM visit will be completed 1–3 months postinsertion. This, combined with formal adjudication of these rhythm strips, will allow for calculation of ICM diagnostic performance.2.5The primary objective of this interim analysis is to characterize the ICM remote monitoring rates and the utilization of the remote programming feature. Future results from LUX‐Dx PERFORM will characterize the performance of the arrhythmia detection algorithms in the LUX‐DX ICM, as well as report the ICM system‐related complication‐free rates, for the full study population, at 30 days 12 months postinsertion.2.6The interim analysis objectives do not have formal statistical hypotheses. Performance of the arrhythmia detection algorithm will be evaluated at the end of the study by calculating the positive predictive value (PPV) of arrhythmias captured by the ICM, using adjudicated HM tracings as the reference standard. ICM system‐related complication‐free rates will be assessed using Kaplan‐Meier methodology comparing the rate and one‐side pointwise log‐log confidence interval with a performance criterion of 94% at 30 days and 93% at 12 months.33.1N = 279) and in‐clinic 23% of the time (N = 83).First enrollment was on March 5, 2021 and as of October 14, 2021, 369 patients were enrolled in the LUX‐Dx PERFORM study and 363 patients had device insertions at 18 centers in the United States  had placed a Tegaderm over the incision site when taking a shower and the skin glue came off when the Tegaderm was removed. The incision was reglued and there were no signs of infection. One patient  returned the day of insertion with incision site bleeding, which resolved with pressure, epi/lidocaine injection, nonabsorbable sutures, and redressing. Three patients had infections ; two were treated with oral antibiotics for infections that occurred ≤10 days post in‐hospital insertion and resolved without further mitigation. The third patient had an infection 50 days post in‐hospital insertion that required treatment with oral antibiotics and ICM explant. There was also one report of ICM erosion and removal , the site was cleaned, and oral antibiotics were ordered for erythema. One patient  developed a rash from a Holter patch that resolved with steroid cream.3.2One concern with using Bluetooth communication as the means of data transfer is that failure to maintain connectivity may occur when mobile devices are out of battery power, or when patients go for extended periods without their mobile device on their person. In this cohort, successful daily data transmission from the ICM to LATITUDE Clarity occurred 86% of the time  and the median percentage of days with a data transmission for individual patients was 94% (interquartile range: 82–99). Failure to maintain connectivity was uncommon and the percentage of patients with at least 1 weekly data transmission remained high over the course of the monitoring period . Details of 13 in‐clinic and 24 remote sessions were reported by the sites ; 92% (33/36) of the changes were applied within 48 h. Two reprogramming sessions using the myLUX Patient app were in progress at the time of data lock and were not included in the analysis.The 3.3.1Two representative examples are presented to illustrate the utility of remote ICM programming.With the first example, an 84‐year‐old gentleman with a prior history of catheter ablation for asymptomatic atrial flutter was referred for an ICM. The patient had been having possible AF episodes according to a commercially available home monitor, but then showed no arrhythmias after a week of monitoring with a wearable cardiac monitor. Post insertion of a LUX‐Dx, a 4‐min‐long “AF” episode was detected with an AF Response setting of “More” and an AF Duration setting of 4 min . Multiple such episodes were detected. These episodes were reviewed by the electrophysiologist and determined to be sinus rhythm with atrial premature beats and sinus arrhythmia Figure . The phyIn the second example, an 84‐year‐old gentleman with hypertension and diabetes underwent LUX‐Dx ICM insertion for the management of asymptomatic paroxysmal AF. The patient was found to have a 5‐h episode  of a regular tachycardia at 120 bpm, suspicious for atrial flutter Figure . The pat4R–R interval were ineffective at detecting atrial flutter and were typically programmed off. Finally, the need for reprogramming to attempt to address these performance issues required an on‐site visit absent the ability to reprogram legacy devices remotely.The utility of ICM placement in the diagnosis and management of arrhythmias has now been verified in a variety of clinical contexts.R–R intervals. Future results from the LUX‐Dx PERFORM study will evaluate the clinical implementation of these technology advances and will characterize quantitative changes in signal quality and arrhythmia detection.The LUX‐Dx ICM was designed to address many of these important issues.ICM system‐related complication‐free rates at 30 days and 12 months postinsertion will also be used to demonstrate acute and chronic safety. The interim results of the LUX‐Dx PERFORM trial presented here suggest a favorable safety profile for the LUX‐Dx ICM. As of this analysis, 363 patients had the LUX‐Dx inserted with few adverse device effects. No infections were reported after in‐office procedures and three patients experienced an infection after in‐hospital insertion procedures for a total infection rate of 0.8% (3/363). Two infections resolved with oral antibiotic treatment, and one resulted in explant of the ICM. This low rate of infection is consistent with other reports of low infection rates (0%–1.6%) The device performance measures reported here affirm in vivo prior findings in an in silico environment. Likewise, transmission rates between the ICM and data management systems have been an issue in the past,The ability to reprogram these devices remotely has the potential to improve workflow, increase throughput, and reduce the need for clinic visits. The interim results presented here already demonstrate that remote ICM reprogramming is feasible and can positively impact arrhythmia detection and diagnosis.The two ICM reprogramming examples demonstrate the importance of signal quality and reprogramming ability. In the first example, the ICM was placed for nonspecific complaints of dizziness, palpitations, and possible AF. After remote reprogramming eliminated most false positives, it became clear that intermittent complete heart block was likely responsible for the patient's symptoms. Rather than employing a rhythm control approach which might have worsened his heart block, proper rhythm information allowed identification and treatment of the actual problem. In the second example, the ability of the AT algorithm to accurately detect atrial flutter in vivo was verified. The ability to remotely program in this setting to improve detection was impactful, unmasking the true burden of atrial flutter and abating tachycardia cardiomyopathy risk.5The presented time periods for reprogramming are real‐world representations of the time from committing programming on the LATITUDE Clarity server to full acceptance and acknowledgment by the device and include circumstantial delays. Examples of such delays include the patient's phone being powered off or with battery depleted, the implanted device being out of range, patients on vacation without phone or service, and so forth.6Interim results from the LUX‐Dx PERFORM study demonstrate the use of remote ICM reprogramming to optimize arrhythmia detection and diagnosis, and to improve clinical workflow. Future results from the LUX‐Dx PERFORM study will characterize improvements in signal quality and arrhythmia detection.Craig Stolen, Brian Kwan, Jonathan Kelly, and David Perschbacher are Employees of Boston Scientific; Jonathan Rosman and John Garner have no conflicts of interest; Harish Manyam is a consultant for Abbott, Boston Scientific, and Jannssen; Mark Richards is the Principal Investigator of the LUX‐Dx PERFORM study and a consultant for Boston Scientific.Supporting information.Click here for additional data file.


Lupus anticoagulant (LA) testing is commonly performed within hemostasis laboratories, and the ACL TOP 50 family of instruments represent a new “single platform” of hemostasis instrumentation. Our aim was to evaluate these instruments and manufacturer reagents or alternatives for utility in LA testing.Comparative evaluations of LA testing using newly installed ACL TOPs 550 and 750 as well as comparative assessments with existing “reference,” predominantly Stago, instrumentation, and reagents. Evaluations comprised both dilute Russell viper venom time (dRVVT) and activated partial thromboplastin time (APTT)‐based assays. Establishment of normal reference ranges (NRR).The HemosIL dRVVT‐based assays showed good comparability with the existing Stago reference method (R > 0.9) and could be considered as verified as fit for purpose. A variety of APTT assays was additionally evaluated for LA utility, and we identified from the assessment good utility of a non‐Werfen solution in Hyphen BioMed Cephen reagents. NRR were established based on ≥120 normal individual plasma samples.This evaluation of LA reagents on ACL TOP 50 Family instruments identified overall acceptable performance of both dRVVT (Werfen solution) and APTT (non‐Werfen solution) to enable harmonization of LA testing in our large network. However, the family is treated as a single instrument class by both the manufacturer  as well as by external quality assessment (EQA) organizations . We recently reported a major evaluation of routine coagulation tests  as performed on the three‐instrument platform, and which formed the basis for a 75 instrument rollout to 60 laboratory sites in our organization, New South Wales (NSW) Health Pathology (NSWHP), being the largest public pathology testing service in Australia.22.1This evaluation was undertaken by NSWHP personnel, and intended to eventually enable accredited implementation of identical methodologies in all NSWHP laboratories performing LA assays. Given LA testing is only performed within the larger sites of our organization, three sites Table  partook The main instruments already in place at the three major evaluation sites were Stago STAR Evolutions Table . An addiEach evaluation site contributed site‐specific data, using a moderate or large number of test samples and a wide range of test results covering both normal and pathological  samples, as available for the evaluation. Samples used were those in excess to test needs, and after diagnostic testing according to the clinical request was completed. All samples were sodium citrate (3.2%) anticoagulated, centrifuged according to local validated centrifugation protocols. Some data related to identical or similar evaluations were also pooled to create a larger “composite” dataset.2.2We also performed several procedures to establish, or verify manufacturer recommended, NRRs for all assays as appropriate. For this purpose, we largely followed the CLSI guidance document “Defining, Establishing, and Verifying Reference Intervals in the Clinical Laboratory,”2.3We primarily evaluated comparative data by linear regression and Bland–Altman difference plots . As noted, some data were also assessed for normality using several statistical tools. Data are otherwise presented numerically or in qualitative synthesis.2.4According to guidance from local Human Research Ethics Committees, formal ethical approval for this evaluation was not sought, as the evaluation represents a Quality Assurance project of method verification using patient samples in excess to needs and which would otherwise be discarded after testing and mandatory short‐term storage according to local accreditation requirements.33.1Key composite data are shown in Figure 3.2APTT patient‐based data for LA testing are shown in Figure 3.3Two sites were able to provided NRR data for both APTT‐ and dRVVT‐based testing on ACL TOPs, and essentially evaluating as per current practice at each site Figure . Both si4We present an evaluation of LA assays (RVVT and APTT based) on the ACL TOP 50 family of instruments  as part of a verification process ahead of planned implementation and accreditation in all diagnostic laboratories that perform such testing and that form part of the largest Public Pathology test service in Australia, namely NSWHP.The evaluations at each site were sometimes similar and at other times different to those of other sites. This was because each site initially had independent separate processes for LA testing, albeit all being based on current guidelines.Some evaluations of ACL TOP instruments have been previously published, as also previously reported by us,There are also very few multicenter studies looking at LA testing, and none that we could locate that was comparable to ours. For example, Moore and colleagues have recently reported a multicenter study centered on Taipan snake venom time as a LA screening test with ecarin time as the confirmatory test.per se, for example based on clinical criteria. Further, we did not specifically assess the influence of anticoagulant therapy on LA test results in our study, with most anticoagulants potentially complicating LA detection and exclusion.We acknowledge several limitations in our study. This study was a laboratory‐based evaluation of new proposed reagents and instruments for use in LA testing, as compared to existing reagents/instruments as reference, which is a requirement of ISO15189 accreditation. That the existing reference was “fit for purpose” was already well established, as successful performance in regular EQA via the local EQA provider, the RCPAQAP.5We essentially verified the utility of the HemosIL dRVVT‐based assay for LA on ACL TOPs and can confirm that these assays have been evaluated as “fit for purpose,” a terminology employed within the ISO15819 standard. We plan to adopt the Cephen reagents for use as paired APTT reagents on the ACL TOPs. Our evaluation identified good performance of these reagents compared to existing methods. Moreover, the APTT reagents represent open methods able to be adapted to a wide variety of instrument platforms, naturally including the ACL TOPs. We also essentially largely verified the manufacturer NRRs for LA testing. We will continue to monitor our LA testing, and performance in EQA, to decide if an additional  study is required to further verify cutoffs in use.We also recognize that accreditation requirements differ according to geographical locality. In Australia, laboratories need to be accredited to ISO 15189 standard. Accreditation of Australian laboratories is overseen by NATA, the National Association of Testing Authorities, and also requires that laboratories adhere to a set of standards as outlined by various guideline documents issued by NPAAC, the National Pathology Accreditation Advisory Council.Finally, this evaluation of LA methods on the ACL TOP 50 Family (550 and 750 in this report) of instruments will enable harmonization of LA testing across all laboratories that perform such testing in our network, being the largest public pathology network in Australia. The ACL TOP 50 Family instruments are now currently installed throughout our network for accredited assays. Universal standard operating procedures (SOPs) and competency and training documents are also in preparation and evaluations for other specialized assays are also in progress.The authors have no conflicts of interest to disclose.Fig S1Click here for additional data file.Fig S2Click here for additional data file.Fig S3Click here for additional data file.Fig S4Click here for additional data file.


As skeletal muscle is one of the largest organs in the body, its damage can directly reflect a decline in somatic function, thus, further affecting daily life and health. Inflammation is a prerequisite for the repair of injured skeletal muscles. Chronic inflammation induced by inadequate repair in skeletal muscle aggravates tissue injury. Exosomes regulate inflammatory responses to facilitate the repair of skeletal muscle injury. Moreover, exosomal miR‐223 with high specificity is the most abundant miRNA in peripheral blood and regarded as biomarkers for inflammation post skeletal muscle injury, which warrants further investigation. Available studies have demonstrated that exosomal miR‐223 negatively correlates with TNF‐α levels in serum and regulates the canonical inflammatory NF‐κB signaling pathway. miR‐223 is a negative feedback regulator with great potential for adjusting inflammatory imbalance and promoting skeletal muscle repair. The research on the regulation of negative feedback factors in the inflammatory signaling pathway is essential in biology and medicine. Therefore, this review mainly elaborates the formation, heterogeneity and markers of exosomes and points out exosomal miR‐223 as a beneficial role in chronic skeletal muscle inflammation and can be expected to be a potential therapeutic target for skeletal muscle damage. Fig 1. Biogenesis and identification of exosomes. Identification of ectosomes and exosomes. (A) Endocytosis and plasma membrane invagination. (B) Formation of early sorting endosomes (ESEs). (C) ESEs give rise to late sorting endosomes(LSEs). (D) LSEs are gradually matured and transformed into multivesicular bodies (MVBs) containing exosomes. (E) MVBs are degraded by lysosomes. (F) The release of exosomes. As one of the largest organs in the human bodyMost cells secrete exosomes, and the functions of exosomes depend on the type of cell from which they originatewww.isev.org/) has paid attention to unifying the name and isolation methods of extracellular vesicles (EVs)Since 2011, the International Society for Extracellular Vesicles  interact with clathrin to form vesicles covered with clathrin through endocytosis and in vagination of the cell membrane. These vesicles after clathrin uncoating are known as early sorting endosomes (ESEs)Exosomes reflecting the source of cells are highly heterogeneous. The heterogeneity of exosomes is manifested with their size, content, functional effect on the recipient cells. Different combinations of these characteristics lead to the complex heterogeneity of exosomes. Proteomic analysis of EVs potentially reveals the heterogeneity of exosomes and reflects the specific proteins derived from parental cellsThe protein, mRNA, and miRNA composition of exosomes are dependent on the parental cell origin. Exosomes can transfer these cargoes to regulate the function of the recipient cell. These biologically active molecules perform a fundamental role in cellular communication and are also a targeted marker to assess the progression and treatment efficacy of diseaseInflammation is defined as a preventive response to stimuli (such as pathogens or damages), the purpose of which is to eliminate stimuli, and to remove dead cells, inducing tissue repair. If controlled, beneficial outcomes are realized, otherwise, the consequences will be devastating if the stimuli persist or the inflammation has not been effectively resolved and become chronic. Typically, inflammation is classified as acute or chronic according to its intensity or duration. Exosome derived therapy emerges as one of the most advanced therapeutic strategies to modulate an overactive immune system. Immune cells actively secrete exosomes after noxious stimulation (such as pathogens or injuries). It has been reported that exosomes could act as either a promoter or inhibitor of inflammation, participating in immune regulationFollowing skeletal muscle injury, macrophages will be activated to polarize and a variety of cytokines are released to result in the inflammatory response. Regarding the process of inflammation after skeletal muscle injury, acute inflammatory damage has long been concerned while chronic inflammatory damage is ignored. In fact, injuryin vivo environment, because dozens of regulatory proteins and their receptors fluctuate rapidly in vivo. Therefore, more critical information about inflammatory cells and their specific inflammatory factors in the process of skeletal muscle regeneration and repair is needed. Exosomes have gradually emerged as messengers of this key information on muscle damageCurrent research on the inflammation after skeletal muscle injury mainly focuses on examining the implication of a single cytokine in skeletal muscle damage. Different respective contents of exosomes can be optimized for treatment at different times of skeletal muscle injury. Although it is the first important step in the investigation of mechanisms underlying inflammation in skeletal muscle, the comprehensive roles of selected cytokines (such as pro‐inflammatory factors) may not be predictable in a more complex Exosomes are cell‐to‐cell communication vesicles that transfer abundant miRNAs across long distances and ubiquitously exist in the circulationIt is reported that miR‐223 can regulate neutrophil activity and enhance macrophage IL‐6 and IL‐1β production. Notably, miR‐223 plays a vital role in inhibiting the development of pro‐inflammatory cellsA large body of evidence supports that miR‐223 is abnormally expressed in patient plasma in influenzaSkeletal muscle macrophages participate in repair and regeneration following injury. Previous studies have reported that macrophages polarize into different phenotypes. It depends on which pathological mechanism of skeletal muscle injury is dominant. Exosomal MiR‐223 involved in the chronic inflammation of skeletal muscle injuryAn existing study has substantiated that the myeloid cells that initially invade into damaged muscle tissues are mainly neutrophils and pro‐inflammatory M1‐macrophages expressing iNOSat the early stages of chronic skeletal muscle inflammation, which clear away the damaged tissueAn in‐depth investigation has revealed the presence of a series of key pro‐inflammatory factors, such as TNF‐α, in the regeneration and repair process after skeletal muscle injury. TNF‐α, a type of cytokine highly expressed by M1‐macrophages, exacerbates muscle damage. After an acute injury, the expression of TNF‐α in the muscle reaches its peak about 24 h after injury, which coincides with the time point when neutrophils and M1‐macrophages invade into the muscle tissues and induce damag.Cell experiments have also confirmed that exosomal miR‐223 could regulate the classical NF‐κB signaling pathway. NF‐κB remained in the cytoplasm in an inactive form after binding to the endogenous inhibitor of exosomal miR‐223. It is unraveled that miR‐223 can down‐regulate TNF‐α in the exosomes induced by inflammation, thereby significantly repressing the inflammatory response and reducing the area of muscle necrosisStaphylococcus aureus. Additionally, restoration of miR‐223 in miR‐223‐deficient (miR‐223‐/Y) neutrophils at the wound potentially improves wound healing, indicating that miR‐223 may guide wound healing in a cell‐autonomous and non‐cell‐autonomous manner, and miR‐223 may inhibit the NF‐κB inflammatory signaling pathway in mouse epithelial cellsMeanwhile, animal experiments confirmed this relationship between exosomal miR‐223 and inflammatory damage as wellExosomes harbor nucleic acids, proteins, lipids and metabolites, making them not only important in cell communication, but also a replacement therapy to treating inflammation. Meanwhile, exosomes are regarded as “the third wagon (secondary to Circulation Tumor Cell (CTC) and Circulating Tumor DNA (ctDNA))” in the field of liquid biopsy secondary to ctDNA and CTC, which could be used as biomarkers of disease progression and treatment efficacy. MiR‐223 is the most abundant miRNA in human peripheral blood microvesicles . As an anti‐inflammatory miRNA of chronic inflammation in skeletal muscle, it is negatively correlated with pro‐inflammatory factors such as TNF‐α in serum. Exosomal miR‐223 also plays an important role in the dysregulated inflammation of skeletal muscle and is expected to be a therapeutic target for chronic inflammation in skeletal muscle.Additionally, miR‐223 is a negative feedback regulator with potentials to address complex chronic condition and is therapeutically viable. Therefore, Further research on more convincing experimental proof on the biological function of miR‐223 to improve clinical efficacy is urgently required. With the progress of molecular biotechnology in the future, it is also still a challenge to accurately regulate the effect of exosomal miR‐223 on target cells.The authors declare that they have no conflicts of interest.


Tribolium castaneum. Specifically, we manipulated yeast supplementation in wheat and whole‐wheat flour in competition assays allowing us to test for sex‐specific effects of food quality (i) on reproductive success and (ii) on the opportunity for selection. Our data show that yeast deprivation in wheat flour had significantly negative effects on body mass and reproductive success of both sexes, while high‐quality flour (whole‐wheat flour) was able to buffer the negative impact to a large extent. Importantly, our data suggest no sex‐specific effect of dietary stress on reproductive success because the magnitude of the negative effect of yeast deprivation was similar for males and females. Moreover, our study demonstrates that low food quality inflated the opportunity for selection and did not differ between sexes neither under benign nor stressful dietary conditions. We discuss the implications of our findings for the adaptation to stressful environments.Environmental factors can have profound effects on the strength and direction of selection and recent studies suggest that such environment‐dependent selection can be sex‐specific. Sexual selection theory predicts that male fitness is more condition dependent compared to female fitness, suggesting that male fitness is more sensitive to environmental stress. However, our knowledge about the effect of environmental factors on sex‐specific reproductive performance and on sex differences in the opportunity for selection is still very limited. In the present study, we investigated the sex‐specific effects of diet quality (yeast deprivation and flour type) in the red flour beetle  Environmental factors can have profound effects on the strength and direction of selection and recent studies suggest that such environmentdependent selection can be sex‐specific. We manipulated diet quality in an insect model finding no sex‐specific effect of dietary stress on reproductive success. Moreover, our study demonstrates that low food quality inflated the opportunity for selection and did not differ between sexes neither under benign nor stressful dietary conditions. They affect reproductive performance and can alter the strength and direction of selection  was found to show sex‐specific diet optima  supplement from the flour mixture that forms the habitat of T. castaneum. Yeast supplementation could be important for red‐flour beetles, as it adds proteins  in popu2Ga1 (wildtype line) and Rd (‘reindeer’ mutant line) used in this experiment had been originally supplied by the U.S. Department of Agriculture and kept in the laboratory for over a year in standard culturing conditions. Rd is a dominant mutation that affects antenna morphology and was used as a paternity marker. All stock beetles were kept in a flour mixture that consisted of organic wheat flour  and 5% dry baker's yeast. In this study we manipulated the diet in two ways: First, we manipulated yeast availability in standard, organic wheat flour , with either control (5% yeast) or no yeast. Secondly, we manipulated yeast availability in organic whole‐wheat flour , with either control (5% yeast), low yeast availability (1% yeast) or no yeast. The low yeast availability treatment was with 1% yeast similar to a previous study  at 50% humidity (±5%) without light. Experimental procedures were performed at room temperature. All flour had been frozen for sterilization prior to experiments at −30°C for at least 24 h. The 2.1In the first assay, we manipulated the diet quality (i.e. yeast availability) of all focal individuals to investigate the effect of dietary stress on reproductive success of males and females in a setup allowing for intra‐sexual competition.Ga1 (wildtype) and Rd adults in each of the treatments. Stock adults were left to mate and oviposit for 3 days in 60 g of flour according to treatment, afterwards the adults were discarded and the eggs were left to hatch. 31 days after the base cultures had been set up, we collected and sexed pupae and established sex‐separated groups of 40 individuals. 8 days after the last pupae had been sexed, all pupae had emerged and were fully adult. We then set up the mating groups for the experiment.We set up base cultures of 80 individuals of Rd), as well as two mating partners. All mating group beetles were synchronized in age (±3 days) and competitors as well as mating partners were always raised on the control diet. All wildtype mating partners were marked with a dot of Revell emaille paint, to differentiate them from the focal. We allowed the treatment groups to mate undisturbed for 3 days in empty arenas . All mating trial arenas were scratched at the bottom to increase traction for the beetles and contained no flour to prevent oviposition. After the mating trial, we froze the males at −30°C for preservation to take phenotypic measurements (see below), while we separated the females to oviposit for 2 weeks in the control environment. In addition, to investigate if females recovered from the treatment when returned to the control diet (i.e. 5% yeast) to lay eggs, we transferred all yeast‐deprived females from the whole‐wheat flour treatment after 1 week to a new egg‐laying vial .Groups contained the focal individual  and a competitor .All frozen individuals from the mating groups were weighed to the nearest 0.01 mg on a Sartorius R200D balance  within 2 months after the main experiment. Body mass was used as a proxy for condition  of all individuals  during development. Hence we mimicked population level dietary manipulation, aiming to measure the opportunity for selection in males and females separately. After the mating trials, all females were given the opportunity to lay eggs on their respective rearing diet (control or yeast deprived). Apart from this, the experimental design followed the procedures described above and was performed in parallel with the first assay.2.3s) as:s represents a standardized measure for the strength of selection against stressed individuals  using the boot package in R  and serves as a proxy for the total net selection . In contrast, in whole‐wheat flour only 0% (control), 0.5% (1% yeast) and 1.1% (no yeast) of females died during the egg laying period .Overall, we observed that development time was considerably longer if yeast was removed from the diet. We did not follow developmental time systematically for all treatments but we collected pupae after 31 and 27 days in the control diet  and this shifted to 48 and 31 days when yeast was removed from the diet. There were no large differences in development time between 5% and 1% yeast in whole‐wheat flour, with the collection of pupae starting after about 29 days in both treatments. In addition, we tracked the survival of females during 2 weeks after eclosion and observed strongly increased mortality when yeast was constantly removed from the diet. In particular, 19% of females died before the end of the egg‐laying period when no yeast was available in wheat flour, which represents a significantly higher mortality compared to 4% of control females that died at the same time . Specifically, in females the mean, proportional reduction in body mass was 8.98%, while in males body mass was only reduced by 4.08% compared to the control. By contrast, yeast availability in whole‐wheat did not influence body mass . In contrast, there was no significant reduction in the offspring number of both sexes in whole‐wheat flour if yeast was reduced or removed from the diet , there we observed significant selection against yeast‐deprived individuals in wheat flour Figure  but no d3.5For males, we were able to estimate the genetic mating success  as a fitness component contributing to the measured reproductive success. In the assay in which only the focal was treated, yeast deprivation had a negative effect on the genetic mating success of males in wheat flour Figure . Neverth3.6I) in T. castaneum in a sex‐specific manner. Therefore, we manipulated the diet quality of all individuals  of the mating groups in a second experimental assay to mimic selection in a population under dietary stress. We measured I as the variance in relative reproductive success of the focal. We found that in this assay, there was a significant effect of the diet treatment on reproductive success of both sexes in wheat as well as whole‐wheat flour  affected the opportunity for selection  body mass, female survival, male mating success and offspring production were negatively impacted by removing yeast from a wheat diet, (ii) the magnitude of the effect of poor diet on offspring production was similar for males and females, (iii) the negative effects of yeast deprivation observed on wheat diet were buffered or undetectable on a whole‐wheat flour diet, and (iv) a poor diet quality inflated the opportunity for selection primarily for females.This study demonstrates that low food quality impairs the reproductive performance of both sexes and affected the opportunity for selection in the red flour beetle 4.1T. castaneum cultured on a low‐quality diet using a non‐nutritional filler , which is in line with a previous study reporting no effect of lowered body mass on fitness in Tribolium also reported a significant increase in development time  was lower in yeast‐deprived individuals that grew on wheat flour. Therefore, the observed effect of yeast availability on male reproductive success might, at least partially, be driven by a lowered competitiveness at pre‐copulatory episodes of sexual selection.Most importantly, we found a negative impact of yeast deprivation on male and female reproductive success in wheat flour. For males, this is in line with the previous finding that investment into spermatogenesis and testes mass was reduced under yeast deprivation in wheat flour  was considerably lower compared to wildtype individuals, which has already been found in previous studies  and studies testing explicitly the effect of pre‐ and post‐copulatory competition on diet‐induced stress responses in males and females. Moreover, the apparent inter‐specific variation in sex‐specific stress responses and its evolutionary link to differences in the strength of sexual selection on males and/or females across species constitutes a promising avenue to follow using comparative approaches.In the context of what is known from other model systems, our findings support the idea that sex‐specific effects of dietary stress on reproductive success can vary across species. In the fruit fly 4.2I) and whether this effect differs between males and females. As predicted, a reduction in food quality increased I in both sexes though this effect was statistically significant only in females under both wheat flour and whole‐wheat flour. This result is in line with the prediction that environmental stress may unmask cryptic genetic variation and therefore inflate I . This suggests that pre‐copulatory sexual selection in terms of male–male competition is higher in yeast‐deprived populations of T. castaneum.The main objective of the second mating assay  was to test for an effect of food quality on the opportunity of selection  is stronger on males ; data curation (lead); formal analysis (lead); investigation (lead); methodology ; resources ; software (lead); visualization (lead); writing – original draft (lead); writing – review and editing . Tim Janicke: Conceptualization ; formal analysis (supporting); funding acquisition (lead); methodology ; project administration (lead); resources ; supervision (lead); visualization (supporting); writing – original draft (supporting); writing – review and editing .Deutsche Forschungsgemeinschaft (DFG) under the grant reference number ‘JA 2653/2–1’. The DFG had no role in the design of the study and collection, analysis, and interpretation of data and in writing the manuscript.This study was funded by the The authors declare that they have no competing interests.


Several categories of epigenetic mechanisms have been studied including chromatin remodeling, DNA methylation, histone modification, and non‐coding RNA mechanisms. These epigenetic changes can have a long‐term effect on gene expression without any underlying changes in the DNA sequences. The underlying pathophysiology of disorders of brain‐gut interaction and stress‐induced visceral pain are not fully understood and the role of epigenetic mechanisms in these disorders are starting to be better understood. Current work is underway to determine how epigenetics plays a role in the neurobiology of patients with chronic visceral pain and heightened visceral nociception. More recently, both animal models and human studies have shown how epigenetic regulation modulates stress‐induced visceral pain. While much more work is needed to fully delineate the mechanistic role of epigenetics in the neurobiology of chronic visceral nociception, the current study by Louwies et al., in Neurogastroenterology and Motility provides additional evidence supporting the involvement of epigenetic alterations in the central nucleus of the amygdala in stress‐induced visceral hypersensitivity in rodents.Epigenetics is a process that alters gene activity or phenotype without any changes in the underlying DNA sequence or genotype. These biological changes may have deleterious effects and can lead to various human diseases. Ongoing research is continuing to illuminate the role of  Our knowledge of the underlying pathophysiology of disorders of gut‐brain interaction (DGBI) such as irritable bowel syndrome (IBS) is still largely unknown.2Stress is a well know adaptive response threats to the body. Many factors modulate an individual's stress response including previous experiences to stressors, gender, ethnicity, hormonal state, and age. The stress response is very complex and includes multiple overlapping and redundant mediators including neuropeptides, steroid hormones, and neurotransmitters.Stress results in an alteration in both synaptic plasticity in the brain and neurotransmission through the hypothalamic–pituitary–adrenal axis (HPA). The HPA axis coordinates both short and long‐term responses to stress through several central mediators including glucorticosteroids and corticotropin‐releasing hormone that modulate behavioral, cellular, and physiological changes and adaptations.3Evidence suggests that early adverse life events are tightly correlated with the initiation and severity of gastrointestinal symptoms in IBS.Stress‐induced pain activation of the sympathoadrenal medullary system also results in release of opioid peptides such as enkephalins from the adrenal medulla. In addition to their utility as measures of the physiological response to pain, neuroendocrine and cardiovascular responses can actively modulate pain perception. Indeed, HPA axis function influences pain sensitivity and some endogenous analgesic responses. For example, in animal models, adrenalectomy increases basal nociceptive sensitivity, and corticosterone replacement increased pain thresholds.The endogenous opioid system in which basal opioid activity may influence pain sensitivity, is one of the most well characterized biological systems involved in the stress‐induced pain response. Higher plasma levels of β‐endorphin are associated with diminished pain sensitivity to stress while elevating β‐endorphin pharmacologically produces increased pain thresholds.Stress alters neuronal activity and neuronal plasticity in several areas of the brain including prefrontal cortex, hippocampus, and the amygdala.4epigenetics in a variety of human diseases and disorders. Of specific interest to the gastrointestinal field, there are many new working models of DBGI, such as IBS, in which epigenetic changes or mechanisms are believed to play an important role in the endophenotypes of patients.Over the last century, we have come a long way in our understanding of epigenetic mechanisms. The term “epigenetics” was first coined by Conrad Waddington, a developmental biologist, in 1942, and over the subsequent 80 years, much work has progressed on the role of DNA methylation in disease states culminating in 2008 with the launch of the NIH Roadmap Epigenetics Program, after which we saw an explosion in knowledge related to epigenetic changes and pathophysiology.Several categories of epigenetic mechanisms that have been studied including DNA methylation, histone modification, chromatin remodeling, and non‐coding RNA. These mechanisms can have a long‐term effect on gene expression without any underlying changes in the DNA sequences. There has been a recent interest in epigenetic regulatory pathways as they play key roles in physiological pathways including regulation of the cell cycle. Since the original studies in 1969 related to its role in long‐term memory, DNA methylation has been one of the most well studied epigenetic changes, particularly over the last 2 decades, in both patients and in animal models.5One of the most widely studied epigenetic mechanisms in visceral nociception is DNA methylation.5′‐C‐phosphate‐G‐3′) sites of GSTM5 (glutathione‐S‐transferases mu 5), SSPO , and TPPP (tubulin polymerization promoting protein) genes in IBS patients compared to controls. These 3 genes are involved in the serotonin system, central nervous system development, brain‐specific proteins, and glutathione and oxidative stress pathways. Further analysis revealed that epigenetic changes in the SSPO genes were positively correlated with anxiety and depression scores in IBS patients.Recent emerging evidence suggests that DNA methylation may also play an important role in patients with DGBI such as IBS, in addition to animal models of visceral nociception. One of the first studies identified genome‐wide DNA methylation changes in peripheral blood mononuclear cells of IBS patients.Cnr1 and Histone acetyltransferases of Trpv1 that lead to decreased expression of the anti‐nociceptive endocannabinoid receptor 1 (CNR1) and increased expression of the pro‐nociceptive endovanilloid (TRPV1) respectively, which induced visceral hyperalgesia. A very interesting finding in this study is that knockdown of DNMT1 and EP300 in rat lumbo‐sacral DRGs decreased DNA methylation and histone acetylation and prevented the stress‐induced increases in visceral pain.Epigenetic changes have also been reported in animal models of stress‐induced visceral hypersensitivity. One such study investigated the epigenetic mechanisms that regulate chronic stress‐induced visceral pain in the peripheral nervous system of rats.Neurogastroenterology and Motility adds further support for the role of the central nucleus of the amygdala leading to stress‐induced visceral hypersensitivity in a rodent model.7 promoter region, a region that is homologous to the human glucocorticoid receptor promoter. Administration of the HDAC inhibitor trichostatin A directly into the central nucleus of the amygdala prevented WAS induced DNA methylation of the glucocorticoid receptor exon 17 promoter region. Overall, WAS increased CpG methylation of the glucocorticoid receptor promoter in the central nucleus of the amygdala in female rats which persisted for up to 28 days.Stress‐induced visceral hypersensitivity in female rats has been shown to be induced by down regulation of the glucocorticoid receptor in the central nucleus of the amygdala.6Substantial evidence exists for the role of epigenetic changes in stress‐induced visceral hypersensitivity in experimental animal models of nociception and in humans with chronic visceral pain. These epigenetic mechanisms may be responsible for the underlying pathophysiology of DBGIs. If future studies can further define the mechanistic role of epigenetics in the neurobiology of chronic visceral nociception, new and more targeted treatment paradigms could be developed for patients suffering from these chronic gastrointestinal symptoms. The end result would then lead to personalized medicine approaches in patients with various endophenotypes of DGBI.Both authors contributed equally to the conception, drafting, and editing of the manuscript.Supported by NIDDK  to GNV & QZ; Department of Veterans Affairs (CX001477‐01) to QZ.The authors have no conflicts of interest to disclose.


Dogs have served as one of the most reliable preclinical models for a variety of diseases and treatments, including stem/progenitor cell transplantation. At the genetic epicenter of dog transplantation models, polymorphic major histocompatibility complex (MHC) genes are most impactful on transplantation success. Among the canine class I and class II genes, DLA‐88 has been best studied in transplantation matching and outcomes, with 129 DLA‐88 alleles identified. In this study we developed and tested a next generation (NGS) sequencing protocol for rapid identification of DLA‐88 genotypes in dogs and compared the workflow and data generated with an established DLA‐88 Sanger sequencing protocol that has been in common prior use for clinical studies. By testing the NGS protocol on a random population of 382 dogs, it was possible to demonstrate superior efficacy based on laboratory execution and overall cost. In addition, NGS proved far more effective at discovering new alleles and detecting multiple alleles associated with gene duplication. A total of 51 new DLA‐88 alleles are reported here. This rate of new allele discovery indicates that a large pool of yet un‐discovered DLA‐88 alleles exists in the domestic dog population. In addition, more than 46% of dogs carried three or more copies of DLA‐88, further emphasizing the need for more sensitive and cost‐effective DLA typing methodology for the dog clinical model. DLA=dog leukocyte antigenFHCC=fred hutchinson cancer centerHLA=HLAHSCT=hematopoietic stem cell transplantationHVR=hypervariable regionIPD‐MHC=immuno‐polymorphism major histocompatibility complex databaseMHC=major histocompatibility complexNGS=next generation sequencingPCR=polymerase chain reaction (Part of EMBL‐EBI)EMBL‐EBI=European Molecular Biology Laboratory European Bioinformatics Institute1The HLA genes in humans and HLA homologs in other mammals play an essential role in immune defense against infection and abnormal cells. They are clustered in the major histocompatibility complex (MHC) and encode transmembrane proteins with a peptide‐binding domain oriented on the exterior surface of the cell. Their primary function is to present an inventory of the antigens from in and around cells to the immune system.Genotyping of HLA class I and II genes is essential for successful hematopoietic cell, tissue, and organ transplantation. These genes have also had longstanding status as markers for disease resistance, autoimmune disorders and, more recently, dysbiosis of the gut microbiome.Dogs provide an informative pre‐clinical model for transplantation of bone marrow stem cells, tissues, and organs.Here we describe an NGS approach to DLA‐88 genotyping. The approach was designed based on an amplicon‐based system for HLA typing22.1Blood samples were obtained from 286 dogs of random breeds from Phoenix Laboratories, Edmonds, WA, as discarded excess diagnostic samples. Samples were identified to us by a unique sample number. The identity of the dog, or the dog owner, was not provided. Genomic DNA was isolated from whole blood using either Puregene DNA purification kit  or QIAamp DNA blood mini kit . This group of dogs included members of 86 distinct breeds; however, it was not possible to accurately assign breed designations to individual animals in this group. For reporting, these samples were treated as a random group of dogs without reference to breed. To supplement the study to include samples where breed designation was assigned, additional DNA samples were obtained from the Cell and Molecular Services and Analysis Lab within the Cooperative Center of Excellence in Hematology (CCEH) at the Fred Hutchinson Cancer Center (FHCC) that were accurately assigned to 48 Great Danes and 48 German Shepherds.2.2Exons 2 and 3 of the DLA‐88 gene, as well as an overlapping region spanning intron 2, were sequenced and phased using a targeted amplicon approach similar to that previously described for the HLA system.www.sciscogenetics.com/pages/kits.html). This system for barcoding with custom primers was adapted from the commercial chimerism kit from Scisco Genetics Inc. (ScisGo®‐HLA‐v5) after exchanging the HLA primer reactions contained in that kit for the DLA‐88 primers. After assembly, the protocol provided for the HLA system was followed precisely using the five DLA‐88 primer sets as five individual amplicons.Amplicons were extended to include barcode sequences (uniquely identifying each genomic sample). The S1 and S2 buffers and the indexing system were supplied by Scisco Genetics Inc., and the basic protocol including PCR reaction cycling was identical to that for HLA typing methods detailed on the product insert for the ScisGo®‐HLA‐v6 typing kit  . Read depths for exons 2 and 3 were greater than 1000 reads per animal whereas exons 2–3 overlapping sequence read depth was on average 225 reads per dog. Database lookup was also used to confirm phase of exons 2 and 3 via comparison of exon sequences with known alleles of DLA‐88 and other class I genes in the IPD DLA database. The system did not attempt to establish typing for DLA‐12. For example, if exon 3 sequences derive from DLA‐88*00402 and exon 2 from DLA‐88*00402  then the database lookup logic will assign DLA‐88*00402 as the DLA‐88 genotype. However, the logic does leave open the possibility that exon 3 is from a known allele, while exon 2 has shown an allele drop off.With target generation and library preparation complete, all 96 bar‐coded samples for each of 5 individual reactions were pooled together into individual tubes and column filtered using a Select‐a‐Size DNA Clean and Concentrator  to remove short (<200 bp) DNA. Filtered pools were quantified using the Quant‐iT™ PicoGreen dsDNA Assay Kit . Quantified pools were combined in equal molar amounts to form a single pool, which was then diluted and denatured in accordance with the standard MiSeq loading protocol . Illumina version 2 chemistry was used for 500‐cycle, paired‐end sequencing. Genotyping was performed using alignment and type selection methods originally developed for HLAAs controls for PCR cross contamination for NGS, each 96‐sample reaction plate contained at least 2 negative wells  and 2 positive samples with known DLA types, previously established by the Sanger method. Appropriately, negative and positive results indicated that the reagents were uncontaminated and functioning as designed. To control for sample‐to‐sample contamination, each reaction plate was assembled in duplicate, and the typing results compared between duplicate samples. Any inconsistency in typing results would be expected to be derived from sample‐to‐sample contamination. All results included in this report were derived from experiments that satisfied these controls.Final allele calls included manual editing of a subset of the samples where the automated processes yielded ambiguous results, and visual inspection was carried out for all results examining read depth and call consistency. All new alleles discovered in this study have been submitted to GenBank and IPD‐MHC with accession numbers as listed in Table 2.3DLA‐88 (exons 2 and 3) was performed as described.Amplification of the polymorphic region of 33.1The canine DLA class I region contains at least three highly homologous DLA class I genes, including DLA‐88, DLA‐12 and DLA‐64, in addition to haplotype variation that includes a duplication of DLA‐88 where DLA‐12 is replaced by a second DLA‐88 locus. This duplicated DLA‐88 has been reported as DLA‐88‐like (DLA‐88L) residing at the neighboring DLA‐12 locus, possibly resulting from a gene conversion event between these genesThis approach was initially tested using a panel of 286 canine DNAs derived from a total of 86 dog breeds, although sample specific breed assignment was uncertain. In order to obtain breed‐specific DLA‐88 frequency NGS typing data, the method was applied to 96 additional samples with assigned breeds consisting of a DNA panel of 48 Great Danes and 48 German Shepherds that had been DLA typed by the Sanger Sequencing method in the FHCC DLA typing shared resource. Results of this analysis are presented in Supplemental Table DLA‐88*072:01 detected in 15 dogs (Table DLA‐88*008:01). A total of 125 unique alleles were detected in this study including 51 new alleles, of which 24 have been officially assigned in the current IPD database with 27 unassigned as of this writing  compared with DLA‐88 alleles or sequences in either the IPD‐DLA database or GenBank. For example, ng Table .A summary of allele calls from NGS analysis is shown in Table The second significant finding was a high rate of new‐allele discovery. A total of 51 unique and previously unknown DLA‐88 allele sequences were detected among the 382 DLA typed samples Table . A measu3.2Selected animals with novel alleles detected by NGS were further characterized for DLA‐88 variation using ABI dye terminator technology (Sanger sequencing) over exons 2 and 3 with phasing deduced from direct sequencing of the PCR products and derivative clones.Direct sequencing of PCR products and deducing alleles from ambiguous sequence traces, failed to identify the majority of the alleles detected by NGS (data not shown). Cloning the PCR product and sequencing eight derived clones (the standard protocol for typing) still missed at least one allele in more than half of the animals, presumably due to allele drop‐off during the PCR. In these cases, the number of sequenced clones was increased to 20 or more.Results of the two methods are compared in Table Even though the Sanger method missed a significant number of alleles detected by NGS, we were able to verify 25 novel alleles based on detection in at least two dogs by both methods  reflecting gene copy number variation, as well as a high discovery frequency of novel DLA‐88 alleles. The NGS method was far more sensitive than Sanger, missing only one call (1.6%) while the Sanger method missed 37% of the allele calls made by NGS , based on the same assumption. The deep sequencing by NGS has a clear advantage over Sanger sequencing in detection of multiple allele dogs. This apparent frequent multiple allele condition in domestic dogs could complicate DLA matching for tissue transplantation. If typing is done by Sanger sequencing, under the assumption of two copies of DLA‐88, vital mismatches could be missed that could lead to unpredicted (and unexplained) rejection.4.3The frequency of new allele discovery was high. A total of 51 novel alleles of DLA‐88 are reported here, 25 of which were verified in at least two dogs by both methods. Since all of the 51 new alleles reported here encode a unique amino acid sequence within the antigen‐binding pocket, they likely represent functionally unique alleles. The rate of new allele discovery in this study , suggests that the real complexity of the DLA‐88 allele set in the canine population is several fold higher than the known complexity so far, consistent with conclusions from prior studies.Although DLA‐88 allelic heterogeneity is evidently greater than previously described, it should be pointed out that it has yet to be established that the new alleles and multiple loci play a role in matching for transplantation. It is possible that some of the new alleles reported are null alleles . The NGS sequencing reported here only provided partial sequencing from genomic DNA, the necessary analysis for full‐length translatable sequences has not been completed, nor has the DLA transcriptome been profiled. While NGS has clear advantages for typing, it's important to point out that matching at the DLA‐88L (or even a third locus) may be excessive for biologically‐irrelevant alleles. Further profiling work to define expression patterns will be needed to improve NGS relevance in the context of transplantation. In that regard, the fact that NGS revealed the existence of potential DLA contributors that the Sanger approach missed, further recommends routine use of NGS in dog transplantation to help drive such studies.Chul‐Woo Pyo: Designed assays and performed NGS sequence analysis, coauthored and edited the manuscript. Michael A. Harkey: Designed and performed conventional sequence analysis, coauthored and edited the manuscript. Beverly Torok‐Storb: Provided scientific and laboratory support and edited the manuscript. Rainer Storb: Designed and initiated the study and edited the manuscript. Alexander Thomas: Performed NGS sequence analysis. Ruihan Wang and Wyatt C. Nelson: Designed computational adaptations for DLA NGS sequence analysis derivative from an existing cloud based and local installation HLA framework. Daniel E. Geraghty: Designed the overall workflow, coordinated laboratory and analysis activities, and coauthored and edited the manuscript.This work was supported by grant U54 DK106829 (BTS) from the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) of the National Institutes of Health (NIH), Bethesda, MD, USA.Daniel E Geraghty is founder and co‐owner of Scisco Genetics Inc. . Chul‐Woo Pyo, Ruihan Wang, and Wyatt C Nelson are employed in part by Scisco Genetics Inc. The remaining authors have no financial relationships that can present a potential conflict of interest in these studies.Appendix S1 Supporting Information.Click here for additional data file.