Phage immunoprecipitation sequencing (PhIP-seq) allows for unbiased, proteome-wide autoantibody discovery across a variety of disease settings, with identification of disease-specific autoantigens providing new insight into previously poorly understood forms of immune dysregulation. Despite several successful implementations of PhIP-seq for autoantigen discovery, including our previous work , current protocols are inherently difficult to scale to accommodate large cohorts of cases and importantly, healthy controls. Here, we develop and validate a high throughput extension of PhIP-seq in various etiologies of autoimmune and inflammatory diseases, including APS1, IPEX, RAG1/2 deficiency, Kawasaki disease (KD), multisystem inflammatory syndrome in children (MIS-C), and finally, mild and severe forms of COVID-19. We demonstrate that these scaled datasets enable machine-learning approaches that result in robust prediction of disease status, as well as the ability to detect both known and novel autoantigens, such as prodynorphin (PDYN) in APS1 patients, and intestinally expressed proteins BEST4 and BTNL8 in IPEX patients. Remarkably, BEST4 antibodies were also found in two patients with RAG1/2 deficiency, one of whom had very early onset IBD. Scaled PhIP-seq examination of both MIS-C and KD demonstrated rare, overlapping antigens, including CGNL1, as well as several strongly enriched putative pneumonia-associated antigens in severe COVID-19, including the endosomal protein EEA1. Together, scaled PhIP-seq provides a valuable tool for broadly assessing both rare and common autoantigen overlap between autoimmune diseases of varying origins and etiologies. Autoantibodies provide critical insight into autoimmunity by informing specific protein targets of an aberrant immune response and serving as predictors of disease. Previously, disease-associated autoantigens have been discovered through candidate-based approaches or by screening tissue specific expression libraries. By contrast, the development of proteome-wide screening approaches has enabled the systematic and unbiased discovery of autoantigens.Two complementary approaches for proteome-wide autoantibody discovery include printed protein arrays and phage-immunoprecipitation sequencing (PhIP-seq) . While pRecently, we and others have adapted and applied PhIP-seq to detect novel, disease-associated autoantibodies targeting autoantigens across a variety of autoimmune contexts . HoweverBeyond the benefits of detecting low-frequency or low-sensitivity antigens, a scaled approach to PhIP-seq would also facilitate increased size of healthy control cohorts. Recently, PhIP-seq has been deployed to explore emerging forms of autoimmune and inflammatory disease, including COVID-19-associated multisystem inflammatory syndrome in children (MIS-C) . HoweverHere, we develop a high-throughput PhIP-seq protocol with markedly increased accessibility (not requiring robotics) and scale , with minimal plate-to-plate variability and low contamination potential, and without sacrificing data quality. We demonstrate the utility of this protocol in the context of an expanded, multi-cohort study, including APS1, patients with immune dysregulation, polyendocrinopathy, X-linked (IPEX), RAG1/2 deficiency with autoimmune features, a KD patient cohort, and emerging COVID-19 patient phenotypes with possible autoimmune underpinnings. In the future, scaled PhIP-seq cohort studies could be used across additional syndromic and sporadic autoimmune diseases to develop an atlas of linear B cell autoantigens.The ability to process large numbers of patient samples for PhIP-seq in a highly uniform manner has several important benefits, including reduction of batch effects between samples from the same cohorts as well as between disease and control cohorts, detection of lower-frequency autoantigens, and the ability to simultaneously include large numbers of control samples.In creating a scaled protocol, we searched for a method that would allow 600–800 samples to be run fully in parallel to reduce any batch or plate-to-plate variability. Thus, each wash or transfer step needed to be performed in rapid succession for all plates. We also prioritized reduction of any well-to-well contamination, particularly given that small, early contamination can amplify across subsequent rounds of immunoprecipitation. Finally, we required our protocol to minimize consumable waste and maximize benchtop accessibility without robotics or other specialized equipment.Escherichia coli lysis without a preceding elution step; shortened lysis step by using square-well plates with semi-permeable membrane for aeration; and options for smaller volume and decreased reagent library preparation in both 96-well and 384-well formats. A detailed protocol, including custom part designs, for both high-throughput vacuum-based and moderate throughput multichannel-based protocols is available at protocols.io.A benchtop vacuum-based protocol  in deep-well 96-well filter plates with single-use foil seals (no well-to-well contamination) met our requirements  or more patients with APS1 . While treviewed inTo improve both sensitivity and specificity, we utilized scaled PhIP-seq to explore expanded cohorts of disease, including a much larger (n=128) APS1 cohort spanning two North American cohorts and one Swedish cohort. All hits present in at least 10% of APS1 patients also spanned all three cohorts, thus further validating broad prevalence of antigens that were previously described by us  and others  . At the Other notable hits with relevant tissue-restricted expression were also observed. For example, SPAG17 is closely related to the known APS1 autoantigen SPAG16 and is expressed primarily in male germ cells and in the lung, with murine genetic mutations resulting in ciliary dyskinesis with pulmonary phenotypes . Also poAPS1 is a clinically heterogeneous disease, and it is also heterogeneous with respect to autoantibodies . BecauseIPEX syndrome is characterized by defective peripheral immune tolerance secondary to impaired T regulatory cell (Treg) function. In IPEX, peripheral tolerance rather than central tolerance is impaired, resulting in a phenotypic constellation of autoimmunity that partially overlaps with APS1 . NotablyA handful of IPEX autoantibodies targeting antigens expressed in the intestine have been described, including harmonin (USH1C) and ANKS4B . In our Several novel putative autoantigens were observed that were shared among three or more IPEX patients . Among tRAG1/2 mutations represent an additional and notoriously phenotypically heterogeneous form of monogenic immune dysregulation. Absent RAG complex activity leads to lack of peripheral T and B cells, therefore causing severe combined immunodeficiency (SCID). However, patients with hypomorphic RAG1/2 have residual capacity to generate T and B cells. Depending on the severity of the defect, these patients can present with Omenn syndrome, atypical SCID (AS), or combined immune deficiency with granulomas and autoimmunity (CID-G/AI) (Hypomorphic ID-G/AI) . AutoimmID-G/AI) .62 patients with hypomorphic RAG1/2 mutations were screened by PhIP-seq to assess for overlap with APS1 and IPEX antigens, as well as for novel autoantigen specificities. Minimal overlap was observed between RAG1/2 deficiency and APS1 and IPEX. However, two samples from RAG1/2 patients indicated the presence of anti-BEST4 antibodies , which wRAG1/2 and/or increased susceptibility to particular infections and will require additional future follow-up.Several other putative antigens among the larger RAG1/2 deficiency cohort were revealed. Nearly half of the cohort sera were enriched for peptides derived from ZNF365 . ZNF365 MIS-C leads to critical illness in ~70% of affected children and shares some common clinical features with KD, the most common cause of acquired pediatric heart disease in the United States. Despite hints for a role of abnormal adaptive immunity and autoantibodies in the pathogenesis of KD and MIS-C, the etiologies of both diseases remain enigmatic . RecentlFirst, 20 MIS-C subjects, 20 pediatric febrile controls, and 20 COVID-19 controls were examined by PhIP-seq, each of which was compared to a cohort of adult healthy controls (n=87). No evidence for specific enrichment was observed for any of the previously reported candidate antigens that overlapped with our PhIP-seq library . MethodoAnalysis of our MIS-C cohort for shared candidate hits yielded only three candidate hits, each in 2/20 patient sera. These were CD34, RPS6KB1, and CAPZB . While tTo screen for possible KD-specific autoantibodies, we analyzed a large cohort of 70 KD subjects by PhIP-seq. KD patients are also often treated with IVIG, so care was taken to ensure that each of these samples was collected prior to IVIG administration. Using the same hit selection criteria as previously, we detected 25 shared hits among 3 or more of the 70 KD samples, which were specific to KD relative to adult healthy controls . Of thesSome of the candidate antigens have possible connections to the systemic inflammation seen in KD, including SPATA2  and ALOX5B . SPATA2 is a protein known to regulate the TNF receptor response, with murine knockout of SPATA2 resulting in increased activation of NFkB and MAPK . SimilarOther KD candidate autoantigens exhibited tissue expression patterns that suggest possible relationships to sub-phenotypes of KD. For example, pancreatitis and psoriasis have been reported as rare complications of KD . We founTaken together, our dataset did not uncover the presence of common autoantibodies in KD or MIS-C. Nonetheless, our findings leave open the possibility of lower-frequency disease-associated autoantibodies, and future studies with increased cohort scaling, diverse representation of clinical subphenotypes, and high-sensitivity follow-up assays may shed further light on the role of autoantibodies in KD.Given the partial clinical overlap of KD with MIS-C, we also searched for the low-frequency, shared KD hits within MIS-C and found that 6 of the 17 KD hits were present in one or more MIS-C samples . Of thesRecently, it was reported that over 10% of severe COVID-19 pneumonia is characterized by the presence of anti-Type I IFN autoantibodies, a specificity that overlaps with APS1 . We therPhIP-seq was then used to investigate patients with severe to critical COVID-19 pneumonia (n=117) relative to patients with mild to moderate COVID-19 (n=55) and non-COVID-19 healthy controls (n=125) . A smallPhIP-seq is a powerful tool for antigen discovery due to its throughput and scalability. Continually declining costs in sequencing paired with scaled protocols such as the ones presented here result in a low per-sample cost and experimental time, and declining costs of custom oligonucleotide based-libraries allow for extensive adaptation . Yet, asOur scaled data reinforces the importance of including large control cohorts to improve specificity and orthogonal validations to evaluate sensitivity. The absence of appropriately sized control cohorts can result in false-positive associations between enriched protein sequences and disease which can lead to misinterpretation. Furthermore, PhIP-seq is optimized for linear antigens and thus is inherently less robust for detection of confirmational or modified epitopes. For example, our previous work highlights that known anti-IFN and anti-GAD65 antibodies can be detected only in a handful of APS1 samples by PhIP-seq, while orthogonal assays using whole conformation protein demonstrate increased sensitivity . SimilarSeveral novel APS1 antigens were identified in this work by virtue of the large cohort size and large control set, enabling the detection of low-frequency hits such as PDYN. PDYN is a secreted opioid precursor of the central nervous system, and while PDYN-knockout mice are largely phenotypically normal, they do experience hyperalgesia and altered anxiety-related behaviors relative to wildtype mice . Future In contrast, antigen overlap between IPEX and RAG-hypomorphic patients was detected in the form of anti-BEST4 antibodies. BEST4 is a well-characterized, cell-surface intestinal ion channel . RecentlAnother novel IPEX antigen, BTNL8, is also of particular interest given its high frequency in IPEX (11/26) as well as its biological functions. BTN and BTNL-family members belong to the family of B7 co-stimulatory receptors known to modulate T-cell responses, with structural similarity to CD80 and PD-L1 . BroadlyWhile some disease cohorts yield strongly enriched common antigens, such as BTNL8 and BEST4, other cohorts appear heterogeneous. Such was the case for MIS-C and KD, where our results suggested only rare, shared putative antigens. Nonetheless, a number of observations point to a possible role for abnormal adaptive immunity and autoantibodies  versus healthy controls (n=186) using the scikit-learn package  with a lAvailable clinical metadata varies across cohorts; all available clinical data is included in North America - 1: All patient cohort data was collected and evaluated at the NIH, and all APECED/ APS1 patients were enrolled in a research study protocol approved by the NIH Institutional Review Board Committee and provided with written informed consent for study participation . All NIH patients gave consent for passive use of their medical record for research purposes. The majority of this human cohort data was previously published Sweden: Serum samples were obtained from Finnish and Swedish patients with APS1. All individuals met the clinical diagnostic criteria for APS1, requiring two of the hallmark components: chronic mucocutaneous candidiasis, hypoparathyroidism, and adrenal insufficiency, or at least one of the hallmark components in siblings or children of APS1 patients. The project was approved by local ethical boards and was performed in accordance with the declarations of Helsinki. All patients and healthy subjects had given their informed consent for participation.North America - 2: All patients underwent informed consent with research study protocols approved by the UCSF Human Research Protection Program (IRB# 10-02467).Patients with RAG1/2 deficiency were enrolled in research study protocols approved by the NIAID, NIH Institutional Review Board . Peripheral blood samples were obtained upon written informed consent.IPEX patient cohort data was collected and evaluated at Seattle Children’s Hospital under an IRB-approved protocol. Clinical data on this human cohort was previously published by UCSD cohort (MIS-C/KD/febrile controls): The study was reviewed and approved by the institutional review board at the University of California, San Diego. Written informed consent from the parents or legal guardians and assent from patients were obtained as appropriate.Rockefeller cohort (MIS-C): All individuals were recruited according to protocols approved by local Institutional Review Boards.All COVID-19 (non-MIS-C) patients were collected between March 01, 2020 and July 21, 2020 and had positive results by SARS-CoV-2 RT-PCR in nasopharyngeal swabs. ZSFG remnant specimens  were approved by the IRB of the University of California, San Francisco. The committee judged that written consent was not required for use of remnant specimens.New York Blood Center & Vitalant Research Institute: Healthy, pre-COVID control plasma were obtained as deidentified samples. These samples were part of retention tubes collected at the time of blood donations from volunteer donors who provided informed consent for their samples to be used for research.Rockefeller cohort: All individuals were recruited according to protocols approved by local Institutional Review Boards. This is an important paper that is methodologically compelling. The work presents a series of enhancements to the PhIP-seq method of autoantibody discovery, improving scaling to larger cohorts and control populations, and increasing the ability to discover disease-specific immune responses. The approach is used to discover a novel, frequent autoantibody response (BTNL8) in IPEX patients, and will be an accessible approach to investigate the presence and specificity of autoantibodies in diseases where these have been difficult to define. public reviews designed to be posted alongside the preprint for the benefit of readers; (ii) feedback on the manuscript for the authors, including requests for revisions, shown below. We also include an acceptance summary that explains what the editors found interesting or important about the work.Our editorial process produces two outputs: (i) Decision letter after peer review:eLife. Your article has been reviewed by 3 peer reviewers, one of whom is a member of our Board of Reviewing Editors, and the evaluation has been overseen by Satyajit Rath as the Senior Editor. The following individual involved in review of your submission has agreed to reveal their identity: Shiv Pillai (Reviewer #1).Thank you for submitting your article "Autoantibody discovery across monogenic, acquired, and COVID19-associated autoimmunity with scalable PhIP-Seq" for consideration by The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission.Essential revisions:1. While all the reviewers recognized that the methodological improvements described have value, there was consensus that they alone do not meet the bar of "substantial developments" or "significant improvements" that a Research Advance require. To move forward there would need to be some application of the new method that reveals important information about an autoimmune disease or an immunological mechanism.Reviewer #1 (Recommendations for the authors):This manuscript may have had two goals – one being to develop methodology for more accurate comparison of autoantibody data across many cohorts with large numbers of controls and the other to gain some information on biological/biomedical relevance.The authors should more lucidly explain how their alterations of the PhIP-seq approach are more than incremental.One of the biological findings of possible note was the shared hits between KD and MIS-C. Perhaps it offers one way in which the disease relevance of this manuscript could be strengthened. This part could be expanded on a bit with more clinical information examining whether the MIS-C patients with antibodies to CGNL1 had more obvious cardiac findings compared to other MIS-C patients and perhaps consider tapping into other MIS-C cohorts for whom clinical information is also available.Reviewer #3 (Recommendations for the authors):It would have been good for the authors to choose another autoimmune process where the expectation of multiple autoantibodies in the 5-30% of patients are known to occur so that they could actually show the method has the ability to find the common autoantibodies. And if it largely cannot, to define the places where it is best applied. The APS1 is a great example, but this does not expand that finding much, and the other examples don't really provide enough new. Essential revisions:1. While all the reviewers recognized that the methodological improvements described have value, there was consensus that they alone do not meet the bar of "substantial developments" or "significant improvements" that a Research Advance require. To move forward there would need to be some application of the new method that reveals important information about an autoimmune disease or an immunological mechanism.We thank the reviewers and reviewing editor for their suggestions. We have responded to each of the points raised by the reviewers in a point-by-point rebuttal below. Given your concerns regarding the importance of our findings, in this revision we include new data in a new independent cohort which confirms the presence of anti-BTNL8 antibodies, a newly found autoantigen in IPEX through an independent radioligand binding assay. These new results offer novel insight into the antigen-specificity of an autoimmune phenotype in IPEX that has remained poorly understood.We address the additional concerns below, in response to the public evaluation summary, highlighting and enumerating the many important novel findings and advances made in this work.Reviewer #1 (Recommendations for the authors):This manuscript may have had two goals – one being to develop methodology for more accurate comparison of autoantibody data across many cohorts with large numbers of controls and the other to gain some information on biological/biomedical relevance.Thank you, we agree with this – the combined goal of our manuscript is to provide a well-characterized PhIP-Seq protocol that enables screening of many human cohorts for providing new findings in biomedical research. We feel that our manuscript demonstrates all 3 of these items, namely (1) a well-validated protocol, (2) application and validation of the approach across many human disease cohorts, and (3) biological relevance through novel, validated autoantigens PDYN, BEST4, BTNL8, and CGNL1, discovered in APS1, IPEX, IPEX/RAG-deficiency, and MIS-C/Kawasaki disease, respectively.The authors should more lucidly explain how their alterations of the PhIP-seq approach are more than incremental.As stated in our introduction, questions of disease-specificity, rare antigens, and antigen overlap can only be addressed in larger, scaled experiments (lines 207-209), which is one of the primary alterations of our protocol. Briefly, most studies to-date have been limited in size, including our own previous work – which, as we demonstrate in this manuscript, both limits the detection of rare antigens and also results in false-positive antigens (lacking disease specificity). The protocol alterations presented here also enable a ~10x larger cohort to be run (in a single experiment), relative to our previous publication, while also ensuring additional advantages including low risk for contamination, benchtop accessibility without requirement for robotics, and low plate-to-plate variability. All of these advances were designed and tested extensively to ensure the highest quality protocol at each step. We have expanded our introduction (lines 210-213) to include an additional instance of description of the advantages and differences in our technique.One of the biological findings of possible note was the shared hits between KD and MIS-C. Perhaps it offers one way in which the disease relevance of this manuscript could be strengthened. This part could be expanded on a bit with more clinical information examining whether the MIS-C patients with antibodies to CGNL1 had more obvious cardiac findings compared to other MIS-C patients and perhaps consider tapping into other MIS-C cohorts for whom clinical information is also available.We thank the reviewer for the thoughtful suggestion. Briefly, for CGNL1, the numbers of positive samples were too low to perform statistical testing. Regarding evaluation of cardiac findings in this these samples, we assume this suggestion is referring to coronary artery aneurysm rather than cardiac dysfunction as CGNL1 is an endothelial antigen and not a myocardial antigen. Qualitatively, the positive samples did not have markedly increased cardiac aneurysm z-scores, with sample number limiting statistical analysis. We agree that future studies looking at new cohorts of MIS-C and KD, particularly those enriched for phenotypes of interest including high cardiac aneurysm z-scores (in KD cohorts) and cardiac dysfunction (in MIS-C cohorts), will be of interest. However, this manuscript (1) is focused on technological advances and (2) already screens and validates antigens with significant clinical relevance in APS1, IPEX, RAG1/2 deficiency, and severe COVID-19. We believe the suggestion to apply the technology presented in this paper to a larger cohort of MIS-C highlights the importance of the advances presented in this paper. Indeed, using PhIP-Seq in significantly larger cohorts of MIS-C cases and controls represents one potential important future application of scaled PhIP-Seq.Reviewer #3 (Recommendations for the authors):It would have been good for the authors to choose another autoimmune process where the expectation of multiple autoantibodies in the 5-30% of patients are known to occur so that they could actually show the method has the ability to find the common autoantibodies. And if it largely cannot, to define the places where it is best applied. The APS1 is a great example, but this does not expand that finding much, and the other examples don't really provide enough new.We were able to find novel antigens which we validated in 15% and 40% of IPEX patients, respectively. Why this finding does not have novelty or substantial value in the absence of known antigens that occur at the requested frequency of 5-30% is unclear to us. This is especially true given that we have demonstrated that in APS1, many of the novel and interesting antigens can be present in a lower percentage of patients even when orthogonally validated.In regard to not providing sufficient novel findings, we respectfully disagree, and have summarized the large number of novel findings above.


In China, low-pass whole-genome sequencing (low-pass WGS) is emerging as an alternative diagnostic test to detect copy number variants (CNVs). This survey aimed to study the laboratory practice, service quality, and case volumes of low-pass WGS-based CNV analysis among national accredited Chinese tertiary hospitals that have routinely applied low-pass WGS for more than a year and that have been certified in next-generation sequencing (NGS) clinical applications for more than three years. The questionnaire focused on (1) the composition of patients’ referral indications for testing and annual case volumes; (2) the capacity of conducting laboratory assays, bioinformatic analyses, and reporting; (3) the sequencing platforms and parameters utilized; and (4) CNV nomenclature in reports. Participants were required to respond based on their routine laboratory practices and data audited in a 12-month period from February 2019 to January 2020. Overall, 24 participants representing 24 tertiary referral hospitals from 21 provincial administrative regions in China returned the questionnaires. Excluding three hospitals routinely applying low-pass WGS for non-invasive prenatal testing (NIPT) only, the analysis only focused on the data submitted by the rest 21 hospitals. These hospitals applied low-pass WGS-based CNV analysis for four primary applications: high-risk pregnancies, spontaneous abortions, couples with adverse pregnancy history, and children with congenital birth defects. The overall estimated annual sample volume was over 36,000 cases. The survey results showed that the most commonly reported detection limit for CNV size (resolution) was 100 kb; however, the sequencing methods utilized by the participants were variable . The diversity was also reflected in the sequencing parameters: the mean read count was 13.75 million reads/case  and the read-length median was 65 bp . To assess further the compliance of the CNV reporting nomenclature according to the 2016 edition of International System for Human Cytogenomics Nomenclature (ISCN 2016), a scoring metric was applied and yielded responses from 19 hospitals; the mean compliance score was 7.79 out of 10 points . Our results indicated that the low-pass WGS-based CNV analysis service is in great demand in China. From a quality control perspective, challenges remain regarding the establishment of standard criteria for low-pass WGS-based CNV analysis and data reporting formats. In summary, the low-pass WGS-based method is becoming a common diagnostic approach, transforming the possibilities for genetic diagnoses for patients in China. PMP22; 601097) in 17p12. In the past decade, chromosomal microarray analysis (CMA) has been adopted as a first-tier test for the detection of clinically significant CNVs [Copy number variants (CNVs) are defined as DNA segments of at least 50 base-pair (bp) in size that present at a variable copy number compared with a representative reference genome . CNVs coant CNVs ,5. Howevant CNVs ,9. Compaant CNVs . Thereupant CNVs ,14. HoweLow-pass WGS-based CNV testing involves three major processes: wet-lab assays , bioinformatic analysis , and reporting  ,16. Eachhttps://www.wenjuan.com/s/nY3MFf2/, accessed on 31 March 2020) and aimed to study the current practices of accredited laboratories, service quality, and case volumes of low-pass WGS-based CNV analysis among Chinese tertiary hospitals. Tertiary hospitals in China refer to a general hospitals that usually have a full complement of services and a higher level of specialized care such as prenatal diagnostic services. It is usually a university-affiliated hospital or women and children’s specialist center, which are equipped with certified prenatal diagnostic laboratories. The inclusion criteria were: Chinese tertiary hospitals that have routinely applied low-pass WGS for more than a year and have been certificated in NGS-based genetic test services for more than three years. For each hospital, one coordinator was assigned as a key informant and was responsible for returning the information from the questionnaires. Participants were required to respond based on their routine diagnostic laboratory practices and data audited in a 12-month period from February 2019 to January 2020. All participants took this opportunity to form the Chinese Genomic Structural Variants Consortium, a working group focusing on improving the quality of service of genome-sequencing-based structural variants detection (including CNVs). In this study, we focused on low-pass WGS for CNVs analysis and did not include the NIPT application. Therefore, hospitals that only applied low-pass WGS for NIPT were excluded from this study.In March 2020, we designed an online questionnaire  for nucleotide variant nomenclature. The type of the variant, band region, genomic coordinates as well as the genome build of these two CNVs were given: DGS heterozygous deletion: 22q11.2; 22:19009792-21452445 (GRCh37), and CMT1 duplication: 17p12; 17:14097915-15470903 (GRCh37).The background characteristics of the departments of the participants as well as the services provided by their departments were investigated in the survey questionnaire. Based on the routine practice of their departments, the composition of patients’ referrals and annual sample volumes of low-pass WGS-based CNV analysis from February 2019 to January 2020 were assessed. Based on the low-pass WGS-based CNV analysis testing provided by the hospitals, participants were required to provide information on the capacity of their hospitals in conducting (1) wet laboratory assays, (2) bioinformatics analyses, and (3) reporting internally; and the sequencing parameters  used. In addition, submissions of a sample report of two representative pathogenic CNVs (DiGeorge Syndrome (DGS) and Charcot–Marie–Tooth Disease type 1A (CMT1A)) were requested to assess their CNV nomenclature compliance with the internationally recognized criteria: ISCN 2016 [Survey data were collected through online questionnaire submissions and further confirmed through phone interviews as well as e-mails for a written record.Overall, 24 Chinese tertiary hospitals  responden = 15,254) were high-risk pregnancies, 33.82%  were spontaneous abortions, 19.66% (n = 7162) were couples with an adverse pregnancy history, and 4.65% (n = 1694) were children with congenital birth defects and suspected genetic disorders, respectively , couples with adverse pregnancy history , high-risk pregnancies , or children with congenital birth defects . In total, there were 36,432 cases subjected to annual testing, of whom 41.87%  , Illumina , and Thermo Fisher Scientific . The platforms utilized are shown in The most frequently reported detection limit of CNV length (resolution) for all primary diagnostic purposes  was 100 In total, 19 participants submitted the reporting nomenclatures for DGS (deletion type of CNV) and CMT1A (duplication type of CNV) based on their routine practice with low-pass WGS-based CNV analysis . Twelve Overall, the compliant score was 7.89 ± 2.04 (mean score ± SD) out of 10 points. Only Hospital 16 received 10 points indicating they used a nomenclature that was fully ISCN 2016 and HGVS-compliant without any errors/inconsistencies assessed in this study. In contrast, only Hospital 3 had a score of 0 because they only provided a simple nomenclature of the affected band of the chromosome and the type of the variant without following the ISCN and HGVS nomenclature format. For the other participating hospitals with scores ranging from 6 to 9, they generally followed the nomenclature format; however, there were elements missing or inappropriately used. Among the hospitals submitting CNV nomenclatures in our survey, 2/19 (10.52%) did not include the symbol of sequencing-based technology, 1/19 (5.26%) did not mention the genome build, 10/19 (52.63%) did not provide the prefix of reference sequences “g.”, 2/19 (10.52%) did not state “del” or “dup” to describe the variant type, and 17/19 (89.47%) contained other errors mentioned previously. All participating hospitals described the affected chromosome as well as the affected chromosome arm and band in their nomenclatures. For the genome build presented in the nomenclature, although “GRCh37” was given as a reference in the questionnaire, more than half of the participants changed it to or added “hg19” in their nomenclature format.In 2019, a joint Chinese expert group’s consensus was published recommending low-pass WGS for clinical application . HoweverOur survey results revealed that low-pass WGS-based CNV analysis was applied as a diagnostic genetic test for high-risk pregnancies, spontaneous abortions, couples with an adverse pregnancy history, and children with congenital birth defects, which affect a significant portion of the population of China ,21,22,23In China, the National Health Commission (NHC) is in charge of the certification of clinical laboratories. Initially, over 100 hospitals were accredited by the NHC in the first batch to conduct molecular testing using NGS technology in 2014 and 17 of our participants were included. The National Centre for Clinical Laboratory (NCCL) is responsible for the external quality assessment (EQA) for nationwide clinical laboratories and healthcare organizations. However, currently, there are no EQA programs for low-pass WGS-based CNV analysis testing. Possible reasons are a lack of standardization and the complex processes involved in low-pass WGS-based CNV analysis, which involves expensive costs associated with manpower from different disciplines, the use of reagents and equipment, and the bioinformatics pipeline and other computing resources. Unlike other straightforward testing, clinical laboratories may use several different approaches when conducting low-pass WGS-based CNV analysis :  and HGVS to assess the compliance level of each description submitted. Our results showed that the mean score of participants was 7.79 , which represented an overall acceptable implementation of the existing gold standard criteria. However, the most common error was the lack of prefix of reference sequences followed by an absence of sequencing technology, variant type, or genome build. These errors resulted in a lack of specificity in the description of a variant and could result in other healthcare providers performing family validation for an incorrect variant, inappropriate interpretation of the clinical significance for the variant, or even providing misleading clinical guidance to patients. Therefore, it is critical to follow the format provided by professional committees. It is worth noting that Hospitals 5, 6, and 7 were using the same platform for low-pass WGS-based CNV analysis and shared the same format and errors, which may indicate that they referenced inappropriate nomenclatures provided by the same software. Therefore, it is important to manually check the automatically generated variant nomenclature in clinical practice. As for the official name for the human genome reference assembly, the Genome Reference Consortium Human Build 37 (abbreviated as GRCh37) is recommended, although it is referred to as hg19 in the UCSC Genome Browser; however, hg19 is not the official assembly’s name or abbreviation.Moreover, the updated edition of the International System for Human Cytogenetic Nomenclature (ISCN 2020), has been published following a thorough revision of the 2016 issue after we conducted this study and it was suggested that all laboratories follow the new 2020 version of criteria starting from April 2020. Therefore, additional key points are to be added to these scoring criteria: the specific genomic reference sequence should be mentioned at the beginning of the HGVS portion; the affected chromosome only needs to be mentioned in the ISCN portion without duplication in the HGVS portion;, and a recommendation to use GRCh38 rather than GRCh37 in the new edition, etc. .In summary, our results indicated that the low-pass WGS-based CNV analysis laboratory service is in great demand in China though it is still in the early stages of its development and requires standardization. From a quality-control perspective, challenges remain regarding the establishment of standard criteria for low-pass WGS-based CNV analysis and data-reporting formats. The low-pass WGS-based method is becoming a common diagnostic approach, transforming the possibilities for genetic diagnoses for patients in China.


Heart transplantation is the gold standard in selected patients with end-stage heart failure. However, immune responses to the transplanted heart can lead to major complications and graft failure, limiting long-term survival. It has been shown that antibodies targeting the heart are elevated in end-stage heart failure patients and before transplantation of the donor heart. We aimed to determine whether elevated antibody levels before heart transplantation are associated with a poor outcome after transplantation. We measured four general subtypes of antibodies  in the blood of patients just before the transplantation was performed. We observe that patients with higher levels of IgG1 and IgG2 antibodies tend to reject the transplanted heart more often compared to patients with lower levels of these antibodies. In addition, we observe that IgG1 and IgG2 levels in these patients remain elevated after transplantation. Together our data suggest that a more active adaptive, antibody-mediated, immune system can lead to graft-related complications in cardiac transplant patients. This implies that a subpopulation of heart transplant patients might benefit from expanded immunosuppressive therapy, which includes medications targeting antibody-producing cells.Cardiac allograft vasculopathy (CAV) and antibody-mediated rejection are immune-mediated, long-term complications that jeopardize graft survival after heart transplantation (HTx). Interestingly, increased plasma levels of immunoglobulins have been found in end-stage heart failure (HF) patients prior to HTx. In this study, we aimed to determine whether increased circulating immunoglobulin levels prior to transplantation are associated with poor post-HTx survival. Pre-and post-HTx plasma samples of 36 cardiac transplant recipient patients were used to determine circulating immunoglobulin levels. In addition, epicardial tissue was collected to determine immunoglobulin deposition in cardiac tissue and assess signs and severity of graft rejection. High levels of IgG1 and IgG2 prior to HTx were associated with a shorter survival post-HTx. Immunoglobulin deposition in cardiac tissue was significantly elevated in patients with a survival of less than 3 years. Patients with high plasma IgG levels pre-HTx also had significantly higher plasma levels after HTx. Furthermore, high pre-HTX levels of IgG1 and IgG2 levels were also significantly increased in patients with inflammatory infiltrate in CAV lesions. Altogether the results of this proof-of-concept study suggest that an activated immune response prior to transplantation negatively affects graft survival. Heart failure (HF) is a complex clinical syndrome in which the heart is functionally and structurally abnormal, which results in reduced cardiac output or increased filling pressures . ApproxiOne of the main risk factors influencing patient survival after transplantation is cardiac allograft vasculopathy (CAV) ,8. CAV iAntibodies against the donor human leukocyte antigen (HLA) are not the only antibodies observed after HTx. Also, antibodies against non-HLA antigens, like angiotensin II type-1 receptor, endothelin receptor type A, and vimentin have been described to be associated with CAV and transplant rejection ,18,19,20In this study, we explored whether pre-HTx antibody levels are associated with post-HTx outcome. We measured immunoglobulin levels at different time points after HTx, in the plasma of the recipient and in post-mortal tissue lysates of the donor heart. We investigated whether there is a correlation between pre-HTx antibody levels and (a) survival post-HTx, (b) and histological CAV stage.The collection of myocardial tissue and blood was approved by the local medical ethics committee of the University Medical Center Utrecht under protocol 12/387 UNRAVEL and in compliance with the Declaration of Helsinki . WrittenThe tissues collected for this study were processed using a standardized procedure . The pron = 61). Tissue lysates of epicardial tissue were made as described previously [All patients whose fresh frozen epicardial tissue was available, including at least 1 of the major coronary arteries were used for tissue lysate antibody analyses  and GraphPad Prism (version 7.04). Two-sided p = 0.01; for IgG2 r = −0.43, p = 0.01). Levels of IgM and IgG3 showed the same correlation, albeit without reaching statistical significance . Since a relatively large proportion of the patients had a survival of less than one month, we performed subgroup analysis in those patients that survived at least one month (n = 21) and investigated whether immunoglobulins levels 1-month post-HTx were similarly associated with post-HTx survival. Indeed, increased levels of IgM, IgG2, and IgG3 1-month post-HTx were also associated with limited post-HTx survival .To investigate whether pre-HTx immunoglobulin levels are associated with post-HTx overall survival, we measured the levels of IgM and IgG1, IgG2, and IgG3, using luminex immunoassay, prior to HTx and compared these levels with survival time post-HTx. High levels of IgG1 and IgG2 correlated significantly with shorter survival post-HTx . Immunoglobulin levels showed the tendency to rise again 6 months after transplantation, returning to levels similar to pre-HTX levels. To assess whether pre-HTx immunoglobulin levels correlate to the immunoglobulin levels observed post-HTx, patients were subdivided into two groups, based on high pre-HTx immunoglobulin levels (above the mean concentration) and low pre-HTx levels (below the mean concentration). Interestingly, patients with high antibody titers prior to transplantation also showed significantly higher immunoglobulin levels in the first-month post-HTx compared to patients with lower pre-HTx levels . In addition, the levels of IgG2 and IgG3 remained elevated 6 months after HTx in patients with high pre-HTx levels compared to low pre-HTx levels (IgG2 p = 0.003 and IgG3 p = 0.0007). This suggests that the immune system of patients with high immunoglobulin levels prior to transplantation and despite the use of immunosuppression remains in a similar degree of heightened activity after transplantation.To get more insight into the alterations in levels of immunoglobulins after transplantation, we measured the immunoglobulin concentrations at different time points after HTx. IgG1, IgG2, and IgG3 levels in the plasma of patients after HTx significantly reduced 1 month after transplantation compared to pre-HTx levels .To study whether local immunoglobulin levels in the heart are also associated with survival, we measured immunoglobulin levels in the epicardial tissue of the transplanted donor heart upon autopsy. Interestingly, in patients that died within the first 3 years after HTx, immunoglobulin levels in epicardial tissue of the donor heart were significantly increased compared to patients that died more than 10.5 years post-HTx . Consistent with high plasma levels, also locally in the epicardial tissue of the donor heart, immunoglobulin levels were significantly increased in patients with H-CAV 1 compared to patients without H-CAV (CAV-0) .Since we observed that levels of IgG1 and IgG2 pre-HTx correlate with post-HTx survival, we hypothesized that elevated immunoglobulin levels in either plasma or tissue lysates might be related to the presence of CAV. An example of histological-CAV phenotype is presented in A strong common denominator both in the progression of HF and post-cardiac transplantation rejection is inflammation . AlthougThough several studies have shown that post-transplantation Ig levels associate with graft survival, we are one of the first to demonstrate a significant correlation between immunoglobulin levels prior to HTx and survival post-HTx, as high levels of IgG1 or IgG2 were associated with a shorter survival time after HTx. Because the patient cohort includes a relatively large group of patients who died within a few days after transplantation the observed association might be influenced by these short-survival patients. Consistently, also in the subgroup analysis of patients that survived at least one month, this correlation was present, as 1-month post-HTx immunoglobulin levels also correlated with survival. This might indicate that high immunoglobulin levels present already before HTx may negatively affect the survival of the cardiac graft. This is in line with the idea of immune sensitization in patients waiting for cardiac transplantation, which was recently highlighted as a major challenge in cardiac transplantation by the American Heart Association and the International Society for Heart and Lung Transplantation . Immune Next, we studied alterations in levels of circulating immunoglobulins from the time of transplantation until 6 months post-HTx. One month after HTx, antibody levels were significantly lower compared to pre-HTx levels. This is likely secondary to the high dose of immunosuppressive therapy given early after transplantation . This isIn addition to plasma, the levels of immunoglobulins were also measured in the epicardial tissue of the donor heart upon autopsy post-HTx. Interestingly, patients who died in the first 3 years post-HTx showed significantly increased levels of immunoglobulins in the transplanted heart compared to patients with a survival of more than 10 years. This observation is in line with the observations in plasma and suggests that an activated adaptive immune system has an adverse effect on graft survival.To assess whether high levels of immunoglobulins pre-HTx may be associated with a certain phenotype of CAV after transplantation, patients were divided into the different H-CAV categories. Increased pre-HTx plasma levels of IgG1 and IgG2 were found in patients with H-CAV 1, the inflammatory phenotype of CAV suggesting active CAV development. In addition, epicardial tissue of the donor heart showed an increase in IgG1, IgG2, and IgM upon autopsy in this CAV 1 type. Previous studies already showed an association between circulating inflammatory markers, such as C-reactive protein (CRP), and CAV development . Our resOur study was primarily designed as a proof-of-concept study and therefore is limited by the relatively small patient group. Therefore, our results need to be confirmed in future studies with larger patient groups. Because our cohort was based on the availability of autopsy, our cohort contained a relatively large percentage of patients with short survival, thereby not reflecting the average survival after transplantation. The cause of CAV development has not been fully clarified. Unfortunately, our study did not allow us to study the mechanistic involvement of autoantibodies in the development of CAV or antibody-mediated rejection, but this is certainly of interest for future studies.In conclusion, in this proof-of-concept study, we show that increased immunoglobulin levels prior to heart transplantation are associated with worse survival post-HTx in a single-center cohort of heart transplant recipients. Interestingly, we show that pre-HTx plasma levels of IgG1 and IgG2 are associated with the inflammatory phenotype of CAV. As the exact cause and mechanisms involved in CAV development are currently not known, our findings may instigate new research into the development and treatment of inflammatory CAV. Overall, our data suggest that a heightened immune activation prior to transplantation may negatively impact graft survival after transplantation. This implies that a subpopulation of HTx patients might benefit from expanded immunosuppressive therapy including B cell targeting therapy, like rituximab. To validate the predictive value of pre-HTX Ig levels for post-HTX survival the results should be established in large-scale cohorts.


The analysis of circulating tumor cell-associated white blood cell (CTC-WBC) clusters represented the progress in the liquid biopsy of malignant tumors, however, related research in patients with colorectal cancer is still absent.To explore associations between CTC-WBC clusters and the prognosis of these patients, we conducted an independent cohort of 329 colorectal cancer patients after curative intent surgery and pre-operative CTC detection in Nanfang Hospital, Southern Medical University, Guangzhou, China between January 1, 2017, and September 31, 2019. The primary cohort referred to patients with CTC-WBC clusters positive. The control cohort was defined as those with exclusively CTCs positive. CTCs were enriched and distinguished by The CanPatrol™ system . The Kaplan–Meier curve was used to compare the progressive-free survival (PFS) and overall survival (OS) between two groups. The COX regression model was used to assess the predictive value of CTC-WBC clusters.P < 0.001) and the rest of clinical characteristics were not markedly associated with the presence of CTC-WBC clusters. Kaplan–Meier curves of PFS and OS exhibited that the CTC-WBC group had significantly shorter PFS (P = 0.011), while not for OS. The multivariate model further suggested that the CTC-WBC clusters  was an independent predictor for the PFS of in post-operation CRC patients.Sixty three patients presented CTC-WBC clusters positive (CTC-WBC group) and 266 patients showed solely CTCs (CTC group). The number of CTCs was significantly different between two groups (The CTC-WBC cluster is significantly associated with recurrence after operation in CRC patients. This finding facilitates the evaluation of this indicator in tumor progression. Colorectal cancer (CRC) has the third-highest rate of estimated new cases and mortality worldwide . The metThe theories concerning tumor metastasis are complex and diverse, one is that circulating tumor cells may travel as individual cells or clusters from the primary site to the microvascular of distant tissues . The cirHowever, it is rather a challenge for tumor cells from the local site to overcome the high pressure and turbulences during blood borne dissemination, as well as the immune attack to seed in distant parenchyma, which makes it difficult for the detection of CTCs in a constant cut-off value . Recent To investigate the prognostic value of CTC-WBC clusters in CRC, we conducted a retrospective, single-center study, comparing the progression free survival (PFS) and overall survival (OS) between the CTC-WBC clusters positive group and the exclusively CTCs positive group in post-operative CRC patients based on a prospectively registered database in China.For this retrospective cohort study, we included an independent cohort of 329 CRC patients in total who had received palliative or radical surgery and CTC detection in Nanfang Hospital, Southern Medical University, Guangzhou, China. Patients were included between January 1, 2017, and September 31, 2019. The blood was drawn from patients for CTC testing approximately 1 week before they received surgery in our hospital and the primary data were collected in the database for later analysis. We constructed the primary cohort with patients who had a confirmed detection of CTC-WBC clusters positive. The control cohort was defined as the exclusively CTCs positive. The study was approved by the ethics review committee of Nanfang Hospital, Southern Medical University, Guangzhou, China. None of the patients were treated with steroids or anti-inflammatory drugs that could affect blood immune cell counts before blood sample collection.The baseline data were obtained from the database of our hospital’s electronic medical record system, including gender, age, histological type, surgery type, depth of invasion, lymph node metastasis, postoperative liver metastasis etc. Blood detection biomarkers included CTC data  and pre-operative CEA, CA199. The disease progression was obtained from our follow-up system, including the disease condition after surgery, the date of recurrence, metastatic or death. Tumor staging was based on the 8th edition of the American Joint Committee on Cancer (AJCC) staging manual . OS was 5 ml peripheral blood samples were collected from CRC patients before surgery in EDTA tubes. CTCs were enriched and distinguished into subgroups by the CanPatrol™ CTC analysis system  within 72 h after intravenous blood drawn. The collecting process was based on nano membrane filtration. The ribonucleic acid in situ hybridization (RNA-ISH) method was adopted to subtype CTCs. The epithelial type was identified using fluorescently labeled antibodies against the epithelial cell adhesion antigen (EpCAM), cytokeratoin 8, 18, and 19. The mesenchymal type was detected by the expression of Vimentin and Twist protein. Leukocyte biomarkers CD45 were used to intercept CTC-WBC clusters. The red fluorescent signal dots represented epithelial-like CTCs and the green dots referred to mesenchymal-like CTCs. The mixed type was observed as a hybrid of red and green dots. CTC-WBC clusters labelled by CD45 were stained as white dots scattered by red, green, or red/green mixture under the automated fluorescence microscope  and R version 4.0.2. The histological type of CRC was classified into nonspecific adenocarcinoma, specific adenocarcinoma  and the mixed. The threshold of CTC number was set according to the median. The baseline data were assessed with the unpaired, 2-tailed Pearson's chi-square tests after the patients were divided into cohorts. The Kaplan–Meier curve was used to compare the prognosis for the CTC-WBC positive group and the exclusively CTC positive group. The Kaplan–Meier curve was checked by the log-rank test. We used the univariate cox proportional hazards analysis to assess the predictive value of CTC-WBC clusters and other indicators. Relevant influencing factors were incorporated in the multivariate cox proportional hazards analysis to compare PFS. In general, 2-sided In this study, a total of 329 post-operation CRC patients were consecutively included. All of them were evaluable and admitted in the final analysis. Among these patients, 218 (66.3%) were men and 111 (33.7%) were women. The age ranged from 16 to 88 and the median age was 58. Most patients have undergone radical surgery , the remaining patients were diagnosed as stage IV and could only receive palliative surgery . The disease severity was staged according to the TNM system, where 41 (12.5%) patients with stage I, 114 (34.7%) patients with stage II, 144 (43.8%) patients with stage III and 30 (9.1%) patients with stage IV. The median number (Inter-quartile Range) of CTCs was 6/5 mL blood (1–116 mL blood), and 176 (53.5%) patients showed the number of CTCs >6. For the type of CTCs, mesenchymal phenotype was expressed in 296 (90.0%) patients. The clinicopathological features classified by CTC-WBC clusters are summarized in Table P < 0.001). We aimed to compare the influence of CTC-WBC clusters on liver metastasis after the surgery, while there is no significant difference between the two (P = 0.274). Next, we investigated whether immune cells induce epithelial-mesenchymal transition feature. Although only 33 (10.0%) patients showed exclusively epithelial CTCs, the CTC-WBC cluster was not correlated with EMT (P = 0.433). The rest of clinical characteristics was not markedly associated with the present of CTC-WBC clusters.Among 329 patients, 63 (19.1%) patients presented CTC-WBC clusters positive (CTC-WBC group) and 266 (80.9%) patients showed solely CTCs (CTC group). The number of CTCs, which divided by the median 6 was significantly different between two groups , preoperative CEA , and CTC-WBC/CTC group  were significantly correlated with the progression free survival (Table P = 0.042), the surgery type  and the pTNM stage  were all independent predictors for the PFS of post-operative CRC patients.  deaths in total and three of them died of non-neoplastic causes. Kaplan–Meier curves of PFS and OS were illustrated to compare the CTC-WBC and CTC group, exhibiting that the CTC-WBC group showed significantly shorter PFS -based approaches had been conducted in colorectal cancer and confirmed the prognostic value of CTC. Later, the CellSearch system (Veridex LLC) was established and became popular , 24. HowThe role of tumor-associated neutrophils (TANs) as a driver of tumor progression have been drilled down deeply by previous studies. Specific examples of transforming growth factor (TGF)-β shaping the pro-tumorigenic (N2) or antitumorigenic (N1) effects of TAN were demonstrated . HoweverAlthough TANs were identified as the supporting factors mediating distant CRC metastasis, especially liver metastasis , the theThe study has several limitations. The significant association with CTC-WBC clusters was neither exhibited in the overall survival, nor the post-operative liver metastasis. Among the primary culprit for causing is the small number of cases from the single-center. Also, a longer follow-up and a consistent CTC monitor after the surgery are needed. The first two factors induce insufficient end-point events, while the latter indicates the lack of pre- and post-operative comparisons and subsequent long-term surveillance. In addition, the post-operative chemotherapy information of patients was not included due to the discharge of patients, which prevented us from adjusting the influence of adjuvant chemotherapy on prognosis or evaluating the therapy efficacy via CTC-WBC clusters. The failure in predicting liver metastasis may be explained by these reasons. Finally, the study was limited by the inherent flaws of retrospective studies and wanted in an extensive exploration of molecular mechanisms as we mentioned above. More work will need to be done to establish multi-center prospective studies with larger-scale samples and a longer follow-up duration.In summary, our study found the presence of CTC-WBC clusters in peripheral blood of preoperative CRC patients. Furthermore, we demonstrated that CTC-WBC cluster at baseline was significantly associated with worse progression-free survival and could serve as an independent predictor for CRC patients undergone radical or palliative surgery, while not for the overall survival. These clinical sample data provide clues for a more convenient and predictive monitoring for CRC patients and indicate a supplementary tool to early identify the occult progression of tumors. The prognosis could be improved by the timely adjustment of chemotherapy and surgery program. It is worth noting that larger-scale, multi-center and prospective studies are needed to obtain more convincing evidence.


Image-guided adaptive brachytherapy (IGABT) using intracavitary and interstitial (IC/IS) techniques plays a pivotal role in definitive radiotherapy for locally advanced cervical cancers. However, the training opportunities for interstitial needle application are limited, preventing this technique from becoming widespread. This study aimed to develop a training simulator for IC/IS brachytherapy. The simulator consists of a soft silicone tumor phantom and acrylic tube mimicking the vagina; it has high visibility because of translucent materials and is compatible with computed tomography (CT) and magnetic resonance imaging (MRI). A patient harboring a typical bulky and irregular-shaped cervical tumor was selected from 495 in-house IGABT-treated candidates, and a tumor phantom (68 × 49 × 45 mm) modeled on this patient was produced from three-dimensional real-scale measurements of the MRI-based high-risk clinical target volume at first brachytherapy. In trial use by two physicians with different levels of IGABT skills, a Fletcher-Suit Asian Pacific applicator, and a Venezia applicator with interstitial needles were nicely applied to the simulator, facilitating successful creation of CT-based treatment plans consistent with clinical practice. Thus, the training simulator can be useful for the training of IC/IS brachytherapy, and warrants further research employing a greater number of phantoms and practitioners to verify its educational value. Cervical cancer causes more than 300,000 deaths worldwide annually, and its mortality ranks fourth among all cancers . RadiothDespite the technological advancements, IGABT using a tandem and ovoid applicator alone results in suboptimal dose coverage for bulky or irregular-shaped tumors, leading to insufficient tumor control ,9. To deThe key to the success of IC/IS brachytherapy is to insert interstitial needles in the locations appropriate to achieving a highly conformal dose to the targeted tumor volume. However, the training opportunities for needle application are limited, thereby preventing this technique from becoming widely used by radiation oncologists. To address this issue, this study aimed to develop a training simulator for IC/IS brachytherapy for locally advanced cervical cancer.The foundation of the simulator was developed using transparent acryl to provide visibility. No metal was used to ensure that the simulator was CT and MRI compatible. To enable a physician to approach through the vagina, an acrylic tube mimicking the vagina (50 mm in diameter and 90 mm in length) was installed on an acrylic plate (200 × 100 mm).A tumor phantom representing a typical bulky and irregular-shaped tumor was produced. The case on which the tumor phantom was modeled was selected by a review of in-house IGABT records. The inclusion criteria for the review were as follows: (i) patients with newly-diagnosed and pathologically-confirmed cervical cancer treated with definitive radiotherapy using IGABT  at GunmaThe HR-CTV data of the model patient were then transferred to a computer-aided design software  and a real-scale 3D volume was exported in acrylonitrile butadiene styrene (ABS) plastic using a 3D printer . An aluminum mold of the ABS plastic tumor phantom was created using a computerized numerical control milling center . Finally, a translucent soft silicon tumor phantom was created using the mold.Trial use of the developed IC/IS training simulator (consisting of a combination of foundation parts and the tumor phantom) was performed by following the clinical flow of IGABT. An expert or resident radiation oncologist performed the application using either a Fletcher-Suit Asian Pacific applicator or a Venezia applicator, with or without interstitial needles. The expert was a Japanese Radiological Society (JRS) board-certified radiation oncologist with 14 years of clinical experience and more than 800 sessions of IC/IS brachytherapy. The resident was a first-grade JRS resident who had experience in approximately 10 sessions of IC/IS brachytherapy. For the sake of simplicity, the number of needles used was fixed at four. No constraints were set on the insertion locations of the needles. With the Fletcher-Suit Asian Pacific applicator, the needles were inserted in a freehand manner, as described previously [192Ir remote-after-loading system  [2cc  below 6 Gy and 7.6 Gy, respectively; these dose constraints are based on the standard protocol for definitive radiotherapy in Japan that employs a central shielding technique for the later 10–20 Gy of EBRT to the whole pelvis [2cc falls broadly within the hard constraints defined in the EMBRACE-II study protocol [90  was utilized as the endpoint of dose coverage to the HR-CTV [After each application, the expert conducted CT-based treatment planning. CT images were obtained using an in-room scanner and the HR-CTV  was delineated using MIM Maestro. Then, the CT images of the model patient used for the actual first brachytherapy were superimposed on those of each trial application, and the contours of the rectum and bladder in the former were reproduced in each trial application. The contour data were transferred to Oncentra  and treatment plans were created based on a high-dose-rate  Sweden) . The hige pelvis ,18, wherIn-house IGABT records of 495 eligible patients were reviewed and 70 stage IIIB patients were identified. Detailed inspection of 3D-volume-rendered data led to the identification of the model patient who harbored the HR-CTV at the first brachytherapy of 68 (left–right) × 49 (anterior–posterior) × 45  mm in size . Using t90 of 4.23 Gy (90 up to 5.69 Gy (by the resident) or 6.70 Gy (by the expert) . Two applicators (a Fletcher-Suit Asian Pacific applicator and a Venezia applicator) were used with or without interstitial needles. The needles were nicely inserted into the tumor phantom via a vagina-mimicking tube with high visibility a,b, foll 4.23 Gy a. The ad expert) b,c. Thes expert) d–f.Despite its efficacy, multiple reports suggest that the use of brachytherapy in cervical cancer treatment has been diminishing in recent decades. In 2013, Han et al. reported a decrease in the brachytherapy utilization rate from 83% in 1988 to 58% in 2009, using data from the National Cancer Institute’s Surveillance, Epidemiology, and End Results program covering 7359 patients with stage IB2 to IVA cervical cancer who were treated with EBRT . More im90 compared with intracavitary brachytherapy alone, with no difference in doses to organs at risk [3 was significantly greater when they were treated with IC/IS brachytherapy. More recently, in a multicenter prospective observational study (EMBRACE-I), IC/IS brachytherapy was used in 43.0% of 1416 cervical cancers, and the overall 5-year local control was 92% [90 greater than 6 Gy regardless of applicator type. It is worthy to note that an ongoing multicenter prospective interventional study (EMBRACE-II) employs stricter dose constraints than those for the previous studies [Accumulating evidence suggests the efficacy of IC/IS brachytherapy for locally advanced cervical cancers. In a large-scale retrospective study (retroEMBRACE), Fokdal et al. reported that IC/IS brachytherapy resulted in significantly greater HR-CTV D at risk . In thei was 92% . Consist studies . These d90 of the plan with freehand needles exceeded that with the Venezia template when the application was performed by the expert. By contrast, the HR-CTV D90 of the Venezia-based plan outperformed the freehand-based plan when the application was performed by the resident. These findings may indicate the presence of applicator- or practitioner-specific caveats when educating practitioners in needle insertion.Optimization of the intratumoral needle location is important for broadening the therapeutic window of IC/IS brachytherapy . FreehanThe training simulator developed in the present study can be easily replicated from the one-off aluminum mold, which will enable inter-practitioner, -modality, and -institute comparisons of IGABT simulation under standardized settings in the future. We can also use this simulator to examine improvements in the performance of the same practitioner or institute. The 3D-tumor printing pipeline reported in this study is applicable to the production of tumor phantoms tailored to individual patients, which could be used in pre-treatment or post-treatment evaluation of IGABT plans in routine clinical practice. Furthermore, 3D-printing technologies can be used in combination with emerging artificial intelligence to improve brachytherapy outcomes .The following limitations to the present study should be noted. First, we developed only one tumor phantom and have reported the results of trial use by only two practitioners, an expert and a resident. The feasibility of the training simulator for educating practitioners in IC/IS brachytherapy should be investigated further by employing multiple phantoms modeled on various irregular-shaped tumors and procedures performed by a greater number of practitioners. Second, we did not assess the dose to the bowel, another organ at risk in IGABT for cervical cancer, and this should be pursued in future studies.To support IC/IS brachytherapy training for bulky and irregular-shaped cervical cancers, we developed a training simulator consisting of a patient-derived soft silicon tumor phantom and an acrylic tube mimicking the vagina. In trial use by two physicians with different levels of IGABT skills, a Fletcher-Suit Asian Pacific applicator, and a Venezia applicator with interstitial needles were nicely applied to the simulator, facilitating successful creation of CT-based treatment plans consistent with clinical practice. Further evaluation employing a greater number of phantoms and practitioners is warranted to confirm its educational value.


Diabetic patients have been severely affected by COVID-19 infection. It has been reported that the disease is more progressive leading to venous and arterial thromboembolism, due to multiple factors. This study was conducted to determine the hematologic parameters including D-dimer in diabetic patients with COVID-19 infection in association with disease severity and treatment. n = 159) from medical records from March to December 2020.  This retrospective cohort study was conducted at King Fahad Medical City, Saudi Arabia, after obtaining IRB approval, by collecting data regarding all laboratory parameters, disease severity, and anticoagulant treatment of COVID-19 diabetic patients , serum fibrinogen (0.007), C-reactive protein (0.005), serum ferritin (0.034), and serum LDH (0.015). Mortality was observed in 14 (8.8%) patients mostly with severe COVID-19 with diabetes. In our study, treatment with low molecular weight heparin was not significantly related to severity. A logistic regression analysis indicated an association of some laboratory parameters with severity and mortality of the disease.  The routine blood parameters if detected early will enable physicians to identify severe cases of COVID-19 patients with Diabetes for prompt treatment and save considerable time and resources. The COVID-19 has already infected more than 643 million people and resulted in more than 6.62 million deaths globally  of 55)  of 55) .μg/mL is the optimal cutoff value of admission D-dimer for predicting mortality in COVID-19 patients, with good sensitivity and specificity suggesting that laboratory interpreters of clinical decline can aid in intensifying the care of the patients with this infection and support in suitable triaging and resource deployment [Bansal et al. showed the significant association of D-dimers with the severity of the COVID-19 disease. Patients with acute lung injury and severe infection will have disseminated intravascular coagulation (DIC) secondary to sepsis . Ayusha A local study also reported that diabetic COVID-19 patients have a higher mortality rate than their nondiabetic counterparts, and patients with uncontrolled diabetes (HbA1C > 7%) on admittance often end up in intensive care . In our p = 0.017 and p = 0.029 among two patient groups, users versus nonusers, respectively [In our study, LMWH was started in 92.5% of all cases. Terpos et al. in a Chinese study reported that LMWH administration among patients with markedly elevated D-dimers or in those meeting the criteria for sepsis-induced DIC was significantly associated with improved 28-day overall survival, ectively .The anti-inflammatory properties of LMWH may be beneficial in COVID-19 patients and should be integrated with other antithrombotic treatments such as antithrombin, and recombinant thrombomodulin to hinder the complex “immunothrombosis” process . In our The association between laboratory parameters and severity of disease has been reported by other studies. Taj et al. in their study concluded that leukocytosis, neutrophilia, elevated neutrophil-to-lymphocyte ratio, APTT, D-dimer, LDH, serum ferritin, and CRP are associated with severity of COVID-19 disease. These laboratory biomarkers therefore play a vital role in the early prediction of the disease .Many infections can be detected using the total blood count test, which can easily be available and cheap but at the same time very informative. Hematological, inflammatory, and other markers can be used to evaluate the severity and prognosis of COVID-19 diseases as there is predictive relevance because all SARS-CoV-2 genetic variants have a significant potential for thrombosis, especially in diabetic patients .The addition of these parameters in the regimens for patients' categorization on admission will facilitate prompt effective intervention and proper decision making in the course of clinical case management .In conclusion, COVID-19 disease affects the hematopoietic system in COVID-19 patients with diabetes leading to major complications and untimely death. This study determines laboratory parameters including D-dimer at admission in diabetic patients with COVID-19 infection in association with disease severity and mortality.Further, optimal anticoagulation regimen with or without adjunct antithrombotic therapies such as LMWH may be useful in patients with COVID-19.Our study has certain limitations. Some patients were excluded because of incomplete documentation of laboratory testing. Our study design is retrospective; hence, findings are limited to the accuracy of record keeping. Furthermore, duration of DM was not recorded which may affect the findings. The results should be interpreted with the caution that diabetes may coexist with other conditions such as cardiac diseases in COVID-19 patients. Therefore, further well-controlled studies are needed in the future to establish an independent role of diabetes in COVID-19.These limitations do not undervalue the findings of the present study which are robust and add value to the limited literature on COVID-19 patients within the Arab region. Further, lack of a control group is another limitation. We suggest multicenter studies to clarify the role of lab parameters in the severity of disease and mortality in COVID-19.


Coronavirus disease 2019 (covid-19), which causes a pandemic in the world, has started to appear in turkey since march 2020. Healthcare workers are at the top of the groups most at risk for covid-19 infection, which can have a negative impact on psychological state.It was aimed to evaluate anxiety and depression levels among healthcare workers.this cross-sectional study performed via an online survey in april 2020. Participants answered questions about sociodemographic features, personal views and experiences about covid-19 and the hospital anxiety and depression scale (hads).A total of 300 healthcare workers,193 men and 107 women, participated in the survey. According to hads, 44.6% of participants scored above anxiety and 68.2% scored above depression cut-off points. Being younger than 50 and taking care of covid-19 patients in hospitals were independently associated with anxiety risk. Female gender, young age (less than 50) and having comorbidity were independent risk factors for depression.Healthcare workers were at high risk of anxiety and depression during covid-19 outbreak. For this reason, psychological support should be given, especially to the group with high risk. On December 2019, some cases of pneumonia of unknown etiology occurred in Wuhan City, China. A new type of virus, named as coronavirus disease 2019 (COVID-19), was found to be the cause of pneumoniaFrom doctors to nurses, all medical health workers are first-line fighters treating patients with COVID-19 Anxiety and depression are common in healthcare professionals due to the stressful working environment and long working hours. More than a fifth of healthcare professionals working in the hospitals screened positive for depression or anxiety ahead of the coronavirus pandemicOutbreaks like COVID-19 not only injure the health, but also have a negative psychological impact on healthcare workers. Previous studies considered severe acute respiratory syndrome (SARS) 2003 outbreak to be a psychological trauma among frontline healthcare workersThere are many factors that may affect the psychological mood in people, such as gender and age. It has been well established that women are in greater risk for anxiety and depression than men, because of the interactions between biological factors and social determinants Therefore, our study aimed to assess mental health outcomes, levels of anxiety and depression, and factors affecting psychological status in healthcare workers during COVID-19 pandemic in Turkey.This is a web-based, cross-sectional study performed via an online survey in April 2020.Between April and May 2020, a total of 300 health workers, 193 males and 107 females, participated in the survey. The study was performed nearly one month after the start of COVID-19 outbreak in Turkey. Data was collected through an online questionnaire using SurveyMonkey software . The web-based questionnaire link was sent to the members of Turkey Respiratory Society, including chest disease specialists, pulmonology research assistants and pulmonology related group members  via e-mail to their personal e-mail addresses. The healthcare workers who joined this survey and answered all the questions were included in the study.The study was approved by Local Ethic Committee of Gazi University School of Medicine, in accordance to the principles in the Declaration of Helsinki.Health care workers who work in Turkey's seven geographical regions participated in the study.The majority of participants were in the 40–49 age group. There were 246 (82%) doctors, 33 (11%) nurses and 21 (7%) other healthcare professionals as participants in our study. Only 17.3% of healthcare workers had at least one comorbidity. The survey included 36 questions with multiple choices. The first 8 questions based on sociodemographic features, such as gender, age and occupation. Questions between 9 and 22 were about COVID-19 experiences (Appendix 1).The anxiety and depression of the study subjects was evaluated using the hospital anxiety and depression scale (HADS), which included questions from 23 to 36Statistical analyses were performed using SPSS version 20.0®. Descriptive statistics were used to present baseline characteristics. Continuous variables were expressed as mean ± standard deviation and median (range). Chi-square test (χ2) was used to compare the differences between groups. Student t test was used to compare the means of 2 independent samples. A P-value < .05 was considered statistically significant. Participants were grouped as individuals with anxiety and depression based on HADS cut-off scores, and binary logistic regression was used to identify factors associated with anxiety and depression. Odds ratio (OR) and 95% confidence interval (95% CI) were obtained from logistic regression models.There were 219 participants (73%) who think that they have sufficient knowledge about Coronavirus. The rate of healthcare professionals who directly followed the coronavirus patient was 73%. The most frequently used protective health equipment was told as the medical mask in contact with the possible / definitive COVID-19 patients (91.2%). Nearly 7% of healthcare workers stated that they received treatment with the diagnosis of COVID-19. While 77.2% of participants expressed feelings of anxiety and fear due to COVID-19, 4% stated that they received psychological support. Participants' mean HADS-A and HADS-D scores were 9.72 ± 5.03 and 9.62 ± 4.32, respectively. HADS total mean score was 19.16 ± 8.84. According to HADS, 44.6% of healthcare workers scored above the anxiety and 68.2% scored above the depression cut-off points. In other words, over two-thirds of healthcare workers were at risk for depression, and nearly half of them had this risk for anxiety.Female healthcare professionals had statistically significant higher HADS-A and HADS-D scores then male . Similarly, participants under 50 years old had significantly higher HADS-A and HADS-D scores when compared with those over 50 . Mean HADS-A scores were statistically higher in the healthcare workers who who took care of COVID-19 patients (p=0.003) and had taken a swab sample from a patient with SARS-CoV2 (p=0.037). There were statistically significant higher HADS-D score in healthcare workers who had at least one comorbidity (p= 0.022). Mean HADS-A and D scores according to characteristics of participants are in Anxiety scores were significantly higher among healthcare workers younger than 50 , women , and individuals taking care of COVID-19 patients , Younger participants (<50), female healthcare workers and individuals with chronic diseases had statistically significant higher depression scores . Being younger than 50  and taking care of COVID-19 patients in hospitals  were independently associated with anxiety risk among healthcare workers. On the other hand, being female , being younger than 50  and having a comorbidity  were predictors for depression among healthcare proffessionals. Table 5 shows the binary logistic regression analysis about the risk factors for depression and anxiety.This web-based study investigated if mental health of Turkish healthcare workers was negatively affected by COVID-19 outbreak. Our results demonstrated a high risk of anxiety and depression in healthcare professionals. Female gender, younger age (less than 50), having an organic disease or a contact with COVID-19 patients were found as potantial risk factors.Healthcare professionals seemed to be at high risk of anxiety and depression during COVID-19 outbreak. The 2019 coronvirus disease is a public health problem that causes international concern and negatively affect the pyschological stateAs all medical health workers are first-line fighters during COVID-19 outbreak, they have a higher risk of exposure to the virus than almost everyone. This fact may disrupt the pyschological stability of the healthcare workers. The main reasons for the psychological distress might be related to the many difficulties, such as feeling of being unsafe at work, the long-term workload at hospitals and the lack of medical protective equipmentIn our study, anxiety and depression levels were found to be higher in women healthcare workers. It may reflect the greater psychiatric impact of COVID-19 pandemic on women. Female gender is vulnerable to depression according to literature dataHealthcare workers younger than age of 50 were found to have high levels of anxiety and depression in our study. Huang et al. similarly reported that younger participants (< 35 years) were more likely to develop anxiety and depressive symptoms during COVID-19 outbreakMinistry of Health have made some arrangements for healthcare workers in Turkey who are on duty during COVID-19 pandemic at the beginning of outbreak. However, due to high anxiety and depression levels detected in our study, we think that some additional precautions should be taken. Providing the best physical, mental, and social conditions may increase confidence and morale of our healthcare workersAnother important topic is preventive measures, such as personal protective equipment. Strengthening prevention and control measures would bring potential psychological benefitsThis study has some limitations. First, the study has a cross-sectional design, which does not allow to make causal inferences. As the survey process included peak time (end of April) of the pandemic in Turkey, this may cause the high level of anxiety and depression among participants. Second, we had no chance to assess the individuals' psychological conditions before COVID-19 outbreak. Third, our study mainly used self-reported questionnaires to measure psychiatric symptoms and did not make clinical diagnosis. A study involving structured clinical interview and functional neuroimaging as the gold standard for establishing psychiatric diagnosiswould give more realistic results.Healthcare professionals were at high risk of anxiety and depression during COVID-19 outbreak. Our report demonstrated potential risk factors as female gender, younger age (less than 50), having a comorbidity and having a contact with COVID-19 patients for healthcare workers to develop anxiety and depression. Therefore, these groups may have priority in terms of psychological support.


Purpose: the pathophysiologic mechanisms explaining differences in clinical outcomes following COVID-19 are not completely described. This study aims to investigate antibody responses in critically ill patients with COVID-19 in relation to inflammation, organ failure and 30-day survival. Methods: All patients with PCR-verified COVID-19 and gave consent, and who were admitted to a tertiary Intensive care unit (ICU) in Sweden during March–September 2020 were included. Demography, repeated blood samples and measures of organ function were collected. Analyses of anti-SARS-CoV-2 antibodies  in plasma were performed and correlated to patient outcome and biomarkers of inflammation and organ failure. Results: A total of 115 patients  were included prospectively. All patients developed severe respiratory dysfunction, and 59% were treated with invasive ventilation. Thirty-day mortality was 22.6% for all included patients. Patients negative for any anti-SARS-CoV-2 antibody in plasma during ICU admission had higher 30-day mortality compared to patients positive for antibodies. Patients positive for IgM had more ICU-, ventilator-, renal replacement therapy- and vasoactive medication-free days. IgA antibody concentrations correlated negatively with both SAPS3 and maximal SOFA-score and IgM-levels correlated negatively with SAPS3. Patients with antibody levels below the detection limit had higher plasma levels of extracellular histones on day 1 and elevated levels of kidney and cardiac biomarkers, but showed no signs of increased inflammation, complement activation or cytokine release. After adjusting for age, positive IgM and IgG antibodies were still associated with increased 30-day survival, with odds ratio (OR) 7.1 (1.5–34.4) and 4.2 (1.1–15.7), respectively. Conclusion: In patients with severe COVID-19 requiring intensive care, a poor antibody response is associated with organ failure, systemic histone release and increased 30-day mortality. The ongoing COVID-19 pandemic, caused by the novel SARS-CoV-2 virus, has caused millions of deaths worldwide and left the healthcare system in many countries in the worst crisis for decades. Since the virus phenotype is novel for humans, no patients have previous antibodies specific for the virus, creating a situation where, in theory, all humans are susceptible for infection and severe disease. Despite this, the clinical course of SARS-CoV-2 infection varies substantially, from asymptomatic carriers to severe multiple organ dysfunction syndrome (MODS) and death, probably explained by individual variations in the immune response.Several risk factors, both for the development of severe disease but also for death, have been identified . Age is Our group previously reported that a weak anti-SARS-CoV-2 antibody response was associated with increased mortality in a small cohort of intensive care patients with COVID-19 disease . The aimThis single centre, prospective observational investigation is a sub-study of the PronMed-study, approved by the Swedish National Ethical Review Agency . Informed consent was obtained either by the patient or by a next-of-kin if the patient was unable to receive information due to their clinical status. The Declaration of Helsinki and its subsequent revisions were followed. The protocol of the study was registered a priori . STROBE guidelines were followed for reporting.All patients admitted to the central intensive care unit at Uppsala University Hospital during the first wave of the pandemic in 2020, with suspected COVID-19 infection, were screened for inclusion.Background characteristics of the patients were obtained through patients’ electronic medical records. Clinical data were collected prospectively daily. Blood samples were taken at ICU admission and three times per week during the time patients were treated in the ICU. Simplified Acute Physiology Score 3 (SAPS3) on admission and daily Sequential Organ Failure Assessment (SOFA) score were calculated prospectively ,28. Acutg for 10 min. After separation, all plasma samples were stored at −80 °C.Peripheral blood from patients with COVID-19 was collected into EDTA- and citrate-containing tubes and plasma was separated using centrifugation at 3000× Complete blood cell counts (CBC), plasma C-reactive protein (CRP), procalcitonin, IL-6, fibrin D-dimer, troponin I and N-terminal pro-brain natriuretic peptide (NT-pro-BNP), kidney function tests (plasma creatinine and cystatin C), liver function tests , aspartate aminotransferase (AST), alkaline phosphatase (ALP)) were performed in the hospital central laboratory. CBC was analysed on a Sysmex XN instrument  while plasma CRP, ferritin, troponin I, kidney and liver markers were analysed on an Architect ci16200 . IL-6 was measured by a commercial sandwich ELISA kit . IgA, IgG and IgM antibodies against SARS-CoV-2 Spike-1 protein were quantified by FluoroEnzymeImmunoassay (FEIA), Phadia AB, Uppsala, Sweden. The analyses were performed on the last sample obtained during the stay at the ICU but within 30 days from symptoms onset to maximise the probability to discover plasma-antibodies. The lower limit of detection was 5 and 20 ug/L for IgA and IgM, respectively, and 10 U/L for IgG.Cytokine and complement analyses are described in detail in the 9 copies/mL and 300 copies/mL as the upper and lower limits of quantitative detection, respectively. The viral RNA analyses were performed at samples taken between day 1 and day 7 in the ICU.SARS-CoV-2 RNA in plasma was determined by reverse transcription qPCR as previously described . For quaThe presence of histones was determined via a semi-quantitative Western blotting method as previously described ,33. In sp < 0.05 was considered significant.Categorical variables are presented as number of observations  and continuous variables as medians and interquartile range (IQR). Comparison between dichotomous variables were made with Pearson’s Chi2-test or Fischer’s exact test as appropriate. Continuous variables were compared with the Mann–Whitney U test. Correlation between antibody levels and SAPS3/SOFA were assessed with Spearman correlation. Analyses of survival in relation to whether patients were positive or negative for antibodies were further assessed with multiple logistic regression while controlling for age. For calculations and figures, SPSS Statistics software, version 23 (IBM) was used. Between 13 March and 28 September 2020, 125 patients were included. After the exclusion of patients without verified COVID-19 infection, patients where no blood samples were obtained and patients where the diagnoses of COVID-19 were considered a secondary finding, 115 patients were included in the final analyses. The vast majority (88%) of the patients in the study were admitted during March–May.The median age for all patients was 62 years and 77% were male . Median Fifty-nine percent of the included patients were treated with mechanical ventilation and 15% received renal replacement therapy. Patients positive for anti-SARS-CoV2 antibodies had higher 30-day survival  compared to patients negative for antibodies . As a complementary analyses, 90-day survival was analysed. This confirmed the findings with higher survival rates in the patient groups positive for anti-SARS-CoV2 antibodies. .Patients positive for IgM also had more ICU-free days, ventilator-free days, renal replacement-free days and vasoactive medication-free days. For IgG and IgA, antibody-positive patients had more renal replacement-free days. In a simple logistic regression model, odds ratios  for 30-day survival were 9.4 (2.6–34.8), 4.0 (1.3–11.6) and 5.6 (1.9–15.9) for patients positive for IgM, IgG and IgA, respectively. When adjusting for age in a multiple logistic regression model, positive tests for IgM and IgG antibodies were still correlated with higher OR for 30-day survival, whereas no difference between the groups were seen for IgA. p = 0.013) and maximal SOFA score . No correlation was seen between IgG and SAPS3 or SOFA whereas the levels of IgM antibodies correlated negatively with SAPS3 .In the correlation analyses, IgA antibody concentrations correlated negatively with both SAPS3  and cardiac biomarkers (NT-proBNP). A prominent activation of the coagulation system with elevated D-dimer and platelet counts is an important feature of severe COVID-19 . HoweverTo further characterise the immune response in relation to antibody positivity, biomarkers of the innate immune system, including cytokines, systemic histone release and complement activation, and white blood cell differential counts were analysed.Antibody-negative patients had significantly higher plasma levels of extracellular histones on day 1 compared to antibody-positive patients, and elevated extracellular histone levels were observed irrespective of the antibody class . We coulComplement factors and activation markers were analysed in plasma samples taken upon ICU admission . There wThe main finding of this study is that a poor antibody response is associated with increased mortality in patients with severe SARS-CoV-2-infection. Furthermore, patients with poor antibody response have higher rates of organ failure based on SAPS3 and SOFA score, even if the correlations are weak and should be interpreted with caution, and also based on the duration of organ support such as renal replacement therapy and vasoactive medication. These clinical associations were corroborated by biomarker analyses, where the strongest associations were seen for kidney function and cardiac biomarkers. In particular, antibody-negative patients had strongly elevated NT-proBNP levels during ICU care, which could indicate an increased rate of circulatory failure in agreement with a trend towards longer duration of vasoactive support in these patients.The strongest signal in our cohort for the associations with outcome is seen for IgM antibodies and it is reasonable to believe that this is due to the natural course of the B-cell development in the germinal centre (GC) reaction. Additionally, IgM, in its pentameric form, has the highest complement-activating capacity among all immunoglobulin subclasses, thus it has a very high neutralising capacity .There was no significant difference in the concentration of viral copies in blood between the analysed groups, although patients negative for antibodies in this study did have numerically higher levels of viral copies in blood. The non-significance might reflect a lack of power in the study, and it would be interesting to analyse this in a larger group of ICU patients. However, we have previously described a lack of strong association between viremia and organ failure in COVID-19 ,36.As the production of antibodies is a result of a close and simultaneous collaboration between the innate and adaptive immune systems, alterations in the innate immune system due to age, gender and/or genetic variations will skew the adaptive responses in different directions . It has This hypothesis is supported by our biomarker analysis, which by several measures indicated a lower degree of inflammation in antibody-negative subjects, possibly due to an unknown inborn or acquired immune defect. Our complement analyses revealed no differences in complement activation between antibody-negative and -positive subjects, but they demonstrated significantly lower circulating levels of intact complement protein C3 and Factor B in antibody-negative patients. These factors are well-known to display an acute phase response pattern and are elevated during systemic inflammation, hence lower plasma levels are agreement with an attenuated inflammatory response in antibody-negative subjects. Furthermore, a recent study on bronkoalveolar lavage in patients with severe COVID-19 describes a more aggravated local immune response in the lung, suggesting plasma analyses might be misleading in compement analyses .Another hypothesis is that patients with a suboptimal adaptive immune response, instead, develop a more powerful non-specific innate immune response, partly supported by previous research , with inOur findings that a poor antibody response is associated with a poor outcome in ICU patients with COVID-19 could have clinical implications. If we could identify patients that are likely to have a slow antibody response early in the disease process, these might be the patients benefiting the most from immune-modulating treatment, as studies, in which patients treated as early as 72 h after COVID-19 diagnosis with convalescent plasma or monoclonal anti-SARS-CoV-2 antibodies, have shown ,50. In aThe strength of this study is the large number of intensive care patients included and the high quality of clinical data prospectively collected in combination with analyses of both antibodies, cytokines, and components of the complement system. The major weakness is that samples for antibody analyses were not taken on the same day as for the other analyses; instead, we chose to analyse the last available blood sample taken in the ICU. The reason for this was to maximise the chance to have samples taken after the patients had developed an antibody response and hopefully also switched antibody class. This might have impacted the results, but there was no difference between the compared groups in median time from admission to sample day.This study confirms that a weak anti-SARS-CoV-2 antibody response in intensive care patients with COVID-19 is associated with increased 30-day mortality and our results suggest that this may be due to multiple organ dysfunction and NETosis.