Minimal residual disease (MRD) refers to a very small number of residual tumor cells in the body during or after treatment, representing the persistence of the tumor and the possibility of clinical progress. Circulating tumor DNA (ctDNA) is a DNA fragment actively secreted by tumor cells or released into the circulatory system during the process of apoptosis or necrosis of tumor cells, which emerging as a non-invasive biomarker to dynamically monitor the therapeutic effect and prediction of recurrence. The feasibility of ctDNA as MRD detection and the revolution in ctDNA-based liquid biopsies provides a potential method for cancer monitoring. In this review, we summarized the main methods of ctDNA detection (PCR-based Sequencing and Next-Generation Sequencing) and their advantages and disadvantages. Additionally, we reviewed the significance of ctDNA analysis to guide the adjuvant therapy and predict the relapse of lung, breast and colon cancer et al. Finally, there are still many challenges of MRD detection, such as lack of standardization, false-negatives or false-positives results make misleading, and the requirement of validation using large independent cohorts to improve clinical outcomes. Liquid biopsy, which has many advantages such as non-invasiveness, acceptability, repeatability and prediction of tumor burden and treatment response, has played an increasingly important role in the diagnosis and treatment of cancer. Cancer biomarkers can be extracted and analyzed from the blood, urine, pleural effusion, seroperitoneum, cerebrospinal fluid or saliva of cancer patients with this novel detection method. Circulating tumor cells (CTCs), cell free nucleic acids, exosomes and other biological components secreted into body fluids by cancer cells are all analytes of liquid biopsies, providing biomarkers such as somatic point mutations, amplifications, deletions, gene fusions, DNA methylation markers, miRNAs, proteins, and metabolites.Cell-free DNA (cfDNA) consists of double-stranded DNA with a length of 150–200 base pairs that circulate mainly in the blood, released through apoptosis, necrosis, and phagocytosis . The oriMRD  is defined as a small number of cancer cells that remain in the body after cancer treatment (those that do not respond to treatment or are resistant to drugs), which may ultimately lead to disease relapse. ctDNA tests can benefit patients with solid tumor for its capacity to confirm the existence of MRD during the postoperative period. On the other hand, MRD tests can monitor and assess the biomarkers that indicate the effectiveness of adjuvant chemotherapy as well as drug resistance . FigureThe ctDNA detection methods are mainly divided into two main categories: the PCR-based detection methods such as Droplet Digital PCR (ddPCR), and the Next-Generation Sequenceing (NGS). These methods have significant differences in detection sensitivity, specificity and coverage.Droplet Digital PCR (ddPCR) is to distribute the DNA sample into millions of water-oil emulsion droplets before the traditional PCR amplification, which means, each of the droplets either contains no gene under test or contains one gene. After PCR amplification, each microdroplet was detected one by one. The initial copy number or concentration of the DNA to be tested could be obtained according to the Poisson distribution principle and the number and proportion of positive microdroplet . The ddPThe detection limit of ddPCR turns out to be about 0.1% . ReporteThe ddPCR method has the advantages of low cost and short detection time, but the disadvantage of detecting only known variants and analyzing only a limited number of variants localize its use in clinical practice as a supplement or conditional substitute for tissue biopsy for genotyping . TherefoNext-generation sequencing (NGS) is a high-throughput technique that enables detection of billions of DNA molecules from a biological sample. Compared with ddPCR method, NGS can search for previously unidentified variations . With thAmplicon-based NGS is one of the popular detection methods to identify specific ctDNA molecules. Gene-specific PCR amplicons are used to amplify the specific genomic regions originated from tumor-derived mutations before NGS is performed. Unique molecular identifiers (UMI) can help increasing sensitivity and specificity of NGS detection . ForshewHybrid capture-based NGS, which hybridize relevant DNA sequences to biotinylated probes before NGS is performed, is developed to improve the detection of multiple mutations of tumor with high sensitivity and without significant prior knowledge .Newman et al. utilized an highly sensitive ctDNA detection technique named Cancer Personalized Profiling by Deep Sequencing (CAPP-Seq) . Cancer The capability of CAPP-Seq includes detection of SNV, insertions/deletion (indel), and genomic rearrangements without individuation. Compared to amplicon-based NGS, Capp-seq shows more reliable detection of copy number changes and allows detection of fusion proteins . Otherwiversus detection limit of detection. Due to the low level of ctDNA in body fluids, targeted approaches, including hybridization capture-based NGS, PCR amplicon-based NGS, are superior to more extensive sequencing approaches such as whole exome or whole genome sequencing.NGS approaches have become prevalent for tumor sequencing and have also been applied to ctDNA detection. WGS applied to cfDNA achieves a sequencing depth of 0.1× and WES achieves a sequencing depth of 100× . AlthougctDNA detection has shown promising clinical potential as a method to detect MRD in solid tumors after radical therapy and before clinical or radiographic disease recurrence . MRD staAmong patients with non-metastatic lung cancer, some patients can be cured by primary surgical resection, radiotherapy and comprehensive treatment including chemotherapy . In factThe TRACERx study showed that MRD was predictive of recurrence before routine imaging and that more than 99% of MRD-negative patients did not relapse after treatment . The timDynamic study prospectively revealed the dynamic changes of ctDNA in patients with primary lung cancer after surgery . After tp < 0.001). The long-term survival rate of patients without ctDNA detected in MRD was significantly higher than that of patients with ctDNA detected (p < 0.001). They suggested that both node-positive and node-negative patients with stage I to III NSCLC may benefit from personalized adjuvant therapy. Patients without tissue material may benefit from tyrosine kinase inhibitors (TKI) or immune checkpoint inhibitors (ICI) with assessing actiable mutations and mutational burden in ctDNA.Chaudhuri et al.  introducp = 0.022). ctDNA-negative patients after chemotherapy had better long-term efficacy than patients with positive ctDNA after chemotherapy .In the study of Kuang’s, they detected tumor tissue-specific mutated ctDNA in preoperative plasma samples from 19 (50%) patients , and preGale et al. reported their study using patient-specific assays with up to 48 amplicons targeting tumour-specific variants unique to each patient to monitor postoperative MRD . Of the A recent study identified a potentially cured population of localized NSCLC by longitudinal MRD detection . From 26In conclusion, MRD detection can identify patients at risk of recurrence earlier and is a practical prognostic factor after radical NSCLC surgery. Positive ctDNA after treatment may indicate the presence of MRD, which may be a signal suggesting a change in treatment regimen. After treatment, ctDNA can change from positive to negative, which means that surgery or adjuvant therapy can remove MRD, thereby changing disease progression and survival.Although tumor biopsy has long been the standard method for tumor detection, its limitations have made minimally invasive and relatively inexpensive liquid biopsy an alternative. For patients with early-stage breast cancer, ctDNA testing can monitor tumor burden and treatment response, so as to guide therapeutic regimen selection.Riva et al. described their study that massively parallel sequencing (MPS) was performed on patients with nonmetastatic triple-negative breast cancer (TNBC) and droplet digital PCR (ddPCR) was used to monitor TP53 mutations expressed in tumor tissues . PatientAnother study used whole exome sequencing to detect mutations in tumor tissue . They thp = 0.012). All patients who achieved pCR were ctDNA negative after neoadjuvant chemotherapy . While ctDNA-positive patients (14%) who failed to achieve pCR (n = 43) showed a significantly high risk of metastatic relapse . 86% of those who did not achieve pCR and had negative ctDNA had a favorable prognosis. The author suggested that even in patients who did not achieve pCR, insufficient ctDNA clearance was an important predictor of poor treatment response and metastatic tumor recurrence, and clearance was associated with improved survival.In the neoadjuvant I-SPY 2 TRIAL, cfDNA was isolated from 291 plasma samples of 84 high-risk early breast cancer patients . 16 patip < 0.05). In addition, they found that ctDNA detection may be a good complement to radiological assessment, due to the median PFS of double DCR group tended to be longer than that of single DCR group .In advanced or metastatic tumors, ctDNA has high clinical value and development prospects because of its relatively high detection rate . RecentlIn the treatment of breast cancer patients, PARP inhibitors are synthetically lethal to TNBC tumors carrying BRCA1/2 aberrations by impairing DNA repair mechanisms . GenomicCompared with the lack of sufficient tumor tissue in the specimen and the need for a long test cycle in the tissue biopsy, the utilization of liquid biopsy to detect ctDNA is expected to become an effective tool to promote precision medicine.For stage II CRC patients, most of them did not receive postoperative chemotherapy. MRD detection is needed to identify 10%–15% of those patients who still have residual lesions after surgery . Postopep < 0.001). For patients received chemotherapy, 15 of 88 (17%) samples were ctDNA positive, with a 30% estimated 3-year RFI. While for those ctDNA undetectable, the 3-year RFI was 77% . The author found out that postoperative ctDNA status was independently associated with RFI and significantly outperformed standard clinicopathologic characteristics as a prognostic marker. They later utilized meta-analysis to summarize their previous studies and concluded that the 5-year recurrence-free rate and overall survival rate of patients with non-metastatic CRC who had detectable ctDNA after surgery were poorer (p = 0.018). This also implies that ctDNA is a better predictor of distant recurrence than local regional recurrence.In a previous study, 40% of patients with stage II colorectal cancer who received 6 months of conventional adjuvant chemotherapy had an absolute risk reduction of only 3%–5%, despite the risk associated with potentially serious adverse events and without means to monitor the efficacy of adjuvant therapy . In anote poorer . In thisp < 0.001). Furthermore, patients received adjuvant chemotherapy had significantly longer 6-month DFS than those who did not .An observational GALAXY study recently analyzed MRD in patients with stage I-Ⅳ colorectal cancer . Within p < 0.0001). Post-nat universal Panel was good at predicting recurrence in patients with high clinical risk, but not in patients with low clinical risk. CNAs analysis also showed a compromised performance in predicting recurrence.In a recent study, Liu et al. used a technique that allows multiple tests of one single cfDNA sample using different methods . They deCurrent methods for monitoring disease status in patients with metastatic colorectal cancer include radiographic imaging techniques and detection of serum CEA levels. However, serum CEA levels may be increased in only 70%–80% of patients .p = 0.003) and longer median PFS  and OS . In another study, the authors used amplicon based deep sequencing to detect ctDNA in mCRC patients  (p = 0.039)   after treatment of many solid tumors . In a st 0.0228) . In anot= 0.039) . Carryin= 0.019) . For rec= 0.019) . In addip = 0.027) and PFS , and the VAF was associated with baseline tumor volume . In addition, patients with residual ctDNA detected after 9 weeks of treatment had worse OS and PFS .In a study of metastatic gastroesophageal cancer, ctDNA was detectable in plasma before treatment in 75% of 72 patients and correlated well with mutations on metastatic biopsy (86% agreement) . The detp < 0.001) and OS . The prognosis of patients with negative ctDNA was better than that of patients with positive ctDNA regardless of tumor stage. In addition, the authors suggested that the combination of ctDNA and AFP detection could improve the prediction performance.A large proportion of the patients with early and intermediate stage liver cancer after surgery will have recurrence. In a recent study, peripheral blood samples were collected from all patients after surgery and analyzed by next-generation sequencing based on hybrid capture . The recThe value of ctDNA in predicting early postoperative tumor recurrence and monitoring tumor burden in patients with hepatocellular carcinoma (HCC) was investigated in another prospective study . They utp < 0.001) were significantly associated with OS. Patients’ CDKN2A mutation in ctDNA  and SMAD4 mutation  were significantly associated with PFS. Disease progression detected by ctDNA was 0.9 months earlier than radiological imaging .Pancreatic cancer is an aggressive solid tumor with a poor prognosis. Currently used biomarkers that are often used to identify advanced pancreatic cancer also do not indicate prognosis. A recent study used hybrid capture-based NGS to sequence ctDNA in patients with metastatic pancreatic cancer . In 40 tp = 0.0148). The hazard ratio for adjusted survival risk increased from 4.13 times to 17.71 times for patients with a risk factor (detectable ctDNA or CA19-9>37 U/ml) compared with patients without risk factors (both p = 0.0055).In another research of patients with borderline resectable pancreatic cancer, no significant decrease in median RFS or OS was observed in ctDNA-positive patients before treatment or after NAC . The medPerioperative systemic chemotherapy can improve the prognosis of upper tract urothelial carcinoma (UTUC). A recent study utilized NGS to analyze perioperative ctDNA to identify patients with poor prognosis who require perioperative chemotherapy . They peIn another study of urothelial carcinoma, the authors improved the performance of the prognostic model by combining ctDNA sequence aggregate VAF (aVAF) values with clinical factors, including age, sex, and liver metastases . In consdoi: 10.1016/j.annonc.2022.05.520. Epub 2022 July 6. PMID: 35809752.When we perform MRD detection, the number of specific variants we focused on was very small because the total number of gene copies in the plasma samples was limited. As we all know, MRD detection often requires a high sequencing depth, the sensitivity of ctDNA analysis is limited, and when VAF lowers close to LOD, the number of specific variants in the sample may be demanding. In addition, the tumor fraction of cfDNA varies between cancer entities and even between patients with the same cancer entity . In someDNA fragments from the clonal hematopoiesis of indeterminate potential (CHIP) or non-neoplastic hematopoietic stem cells can lead to false-positive ctDNA results, which can be reduced by advanced bioinformatics analysis or comparison of ctDNA sequencing with leukocytes and/or matched tumor tissue . These mAgreement between ctDNA and tissue-based NGS results is typically defined as the presence or absence of identical genomic alterations in a single gene on both molecular platforms. The main reasons for inconsistent blood and tissue detection are biopsy location and time, different DNA shedding, tumor heterogeneity, and epigenetic modifications. Lack of standardization between ctDNA tests is another barrier, which limits the understanding of the available results. Inconsistent ctDNA results may be the result of several variables, including the time of sample collection, sample collection process, sample storage method, library construction process, utilization of unique molecular identifiers and bioinformatic analysis.Accurate risk assessment and adjuvant therapy are very important for cancer patients. ctDNA testing can accurately identify the MRD after primary tumor resection, and thus identify the patient population that needs further adjuvant chemotherapy, so as to avoid unnecessary additional treatment. In addition, determining the duration of adjuvant therapy based on ctDNA clearance can help reduce adverse reactions. However, many researchers also suggested that adjuvant therapy based on negative ctDNA testing should not be excluded due to the low standardization of ctDNA detection procedures and the limitations of ctDNA testing techniques.Although preliminary data on the clinical application of ctDNA in MRD detection is promising, most of the studies that provide evidence to support it are small, limited in scope and require validation using large independent cohorts . It is oOverall, MRD aids in the management of cancer at all stages, including screening, guiding adjuvant treatment, predicting relapse early, initiating systemic treatment and monitoring response, and genotyping resistance. Liquid biopsy, espesially ctDNA, can be used as an alternative to tumor tissue detection, especially when tissue biopsy is not feasible or time does not permit. New technologies are being developed, such as methylation pattern-based sequencing which have the potential to optimize ctDNA detection for use in a wide range of scenarios. In the future, we need to carry out more intervention studies to provide stronger evidence support for the application of MRD detection methods, so as to achieve the purpose of integration with clinical routine. Through the monitoring of ctDNA, the therapeutic regimen can be adjusted in time, and the treatment effect can be improved to maximize the survival time of patients.


Historically, much of the discussion surrounding infant feeding focused on promoting breastfeeding the first six months (at least) of an infant’s life, and avoiding breastmilk substitutes. In 1981, the World Health Organization (WHO) developed the International Code of Marketing of Breast-Milk Substitutes to restrict the marketing of breastmilk substitutes.The world is experiencing a continued global transition towards increasing consumption of infant formula, growing-up formula and processed baby foods:,This increased consumption represents a critical change in infant and toddler food habits as historically, food habits were centred around minimally processed food. The shift towards feeding infants and toddlers ultra-processed foods brings health risks such as increases in abdominal obesity and worse future health outcomes, and may impact future food preferences.A key shift in the infant and toddler food sector is in the amount of sugar added to these foods. Excess sugar consumption is a primary cause of obesity and related diseases, including diabetes, heart disease and some cancers.Here we use data from Euromonitor InternationalBetween 2010 and 2022, global sales in the infant and toddler feeding sector overall grew from US$ 33.2 billion to US$ 67.9 billion . Infant The global baby food market has grown from US$ 9.6 billion in 2010 to US$ 17.9 billion in 2022 . EmerginAn increase in sales of sugar through the infant and toddler food sector took place, from 697 billion grams in 2010 to 1009 billion grams in 2021 . ResearcThe increase in availability and variety of baby foods comes at a time when we are learning that the consumption of ultra-processed foods containing multiple ingredients and additives poses health risks. Taste preferences and dietary habits formed during early childhood can persist into adulthood. The diets of infants and toddlers in the United States and across high-income countries are dominated by ultra-processed foods.The data presented here raise major questions about the role infant and toddler food companies play in rising global obesity levels and the double burden of malnutrition in low- and middle-income countries. Today every country has more than one fourth of their adult population overweight and not a single country has seen a significant decrease in adult overweight and obesity in recent decades.Given the increasing evidence base demonstrating the adverse long-term health effects of consumption of ultra-processed foods and added sugars, global concerns now exist for infant and child health. By definition, milk formulas themselves are ultra-processed foods, typically consisting of powdered milk proteins, vegetable oils, lactose and other added sugars, micronutrients and additives. The five largest global formula companies are Nestlé, Danone, Abbott, RFC and RBMJ with four out of these five companies present in more than 100 countries.The diets of infants and young children worldwide are undoubtedly becoming increasingly highly processed, a trend mirrored by increased global consumption of ultra-processed foods. Policy-makers need to ensure these products and the companies who manufacture them are monitored closely, and marketing of these products is more tightly regulated. Future research should investigate options for regulatory measures that could address products that currently lie outside the scope of the code. Doing so will be of critical importance in low- and middle-income countries, where socioeconomic factors and the broader food environment put vulnerable populations at risk.


Thromboelastography (TEG) informs the need for blood product transfusions to prevent procedural bleeding complications in patients with cirrhosis. We aimed to evaluate the impact of using a TEG-based transfusion protocol on blood product utilization before paracentesis and the post-paracentesis hemoperitoneum (PPH) incidence.r value > 10 min or MA <30 mm. Patients were divided into pre-TEG and post-TEG protocol cohorts based on the date of paracentesis. Pre-paracentesis blood product transfusions in the form of platelets, fresh frozen plasma, and cryoprecipitates were recorded. PPH was defined as a decrease in hemoglobin of ≥1 g and the presence of blood on diagnostic imaging and/or the need for therapeutic intervention.We conducted an ambispective analysis of patients with cirrhosis who underwent paracentesis from 2017 to 2021. In May 2019, we enacted a TEG-based transfusion protocol to guide pre-paracentesis blood product use. Patients with platelets < 20,000 or international normalized ratio ≥ 4 underwent TEG and received blood products if p<0.001) with associated cost savings. One patient in each cohort developed PPH.A total of 483 patients underwent 1281 paracenteses. The main etiologies of cirrhosis were alcohol (43%) and NASH (25%), and the mean MELD-sodium was 22±6. Pre-TEG and post-TEG protocol cohort sizes were similar: 253 patients and 607 paracenteses versus 230 patients and 674 paracenteses. After TEG-protocol implementation, blood product transfusions decreased significantly (228 vs. 49 products, Implementation of a pre-paracentesis TEG-based transfusion protocol for patients with cirrhosis successfully resulted in decreased blood product use with no associated increase in incidence of PPH.  Nevertheless, these conventional parameters have often driven the use of blood transfusions despite limited evidence of clinical benefit.5The risk of procedural bleeding in patients with cirrhosis can be difficult to determine due to concurrent thrombocytopenia and coagulopathy. Historically, patients with advanced liver disease were presumed to have a higher risk of periprocedural bleeding based on abnormal levels of standard laboratory parameters, including low platelets (PLT) and elevated INR. However, further studies have demonstrated that cirrhosis constitutes a state of “rebalanced” coagulation profile. Contributing factors to this distinct coagulation profile include diminished hepatic synthesis of clotting factors and reduced PLT secondary to splenic sequestration, which are offset by a reduction of anticoagulant proteins and preserved generation of thrombin.6 Per randomized controlled trials, the use of TEG in patients with cirrhosis has been associated with the safe and effective utilization of blood products.9 More specifically, these trials have demonstrated considerably lower use of blood products when based on TEG values as opposed to conventional parameters . We aimed to evaluate the impact of a TEG-based transfusion protocol on blood product utilization before paracentesis and the incidence of post-paracentesis hemoperitoneum (PPH).Thromboelastography (TEG) is a point-of-care test that gives a global assessment of hemostatic function (clot formation and lysis) reflected in real time.In this ambispective analysis, we evaluated adult patients with cirrhosis who underwent paracentesis at the University of Pittsburgh Medical Center from 2017 to 2021. Data were retrieved from the University of Pittsburgh Medical Center’s secure electronic medical record. We included patients ≥ 18 years old with established cirrhosis. The diagnosis of cirrhosis was based on clinical presentation, diagnostic imaging, or prior liver biopsy. Exclusion criteria included patients under the age of 18 years and patients with any evidence of active bleeding at the time of paracentesis. All patients and/or their medical decision-makers consented to paracentesis. Paracentesis was performed in either an inpatient or outpatient setting, and all were performed under ultrasound guidance per institutional protocol.Patients were divided into pre-TEG and post-TEG protocol cohorts based on the date of paracentesis with respect to the time of protocol implementation (June–July 2019). All patients were followed clinically for the next 48 hours for the onset of PPH, including daily hemoglobin checks. PPH was defined as a drop in hemoglobin ≥ 1 gram plus one or more of the following: the presence of i.p. bleeding on diagnostic abdominal imaging or the need for therapeutic intervention . Our primary outcome was the difference in the total number of blood products transfused  between the pre-TEG and post-TEG protocol cohorts. Secondary outcomes were the onset of PPH within 48 hours after paracentesis and direct health care costs attributed to blood product use and TEG processing in the 2 cohorts, respectively.11r value: A prolonged r value (>10 min) reflects a quantitative or qualitative deficiency of coagulation factors, which may be corrected by plasma transfusion, prothrombin complex, or anticoagulant reversal.Maximum amplitude (MA): A low MA (<30 mm) indicates a quantitative or functional deficiency of PLT and could be corrected by platelet concentrate transfusion.TEG is a point-of-care, global, hemostatic tool that evaluates the dynamics of clot formation. It provides real-time data on clot strength and stability between soluble clotting factors and inhibitors and PLT Figure . In this14 Patients meeting clinical criteria for higher bleeding risk underwent TEG to determine if any preprocedural blood product transfusion was warranted. Recommendations, however, did not apply to patients on anticoagulation or antiplatelet therapy other than aspirin. There was also a consideration for desmopressin and tranexamic acid for conditions associated with platelet or coagulation dysfunction.15 The protocol also instructed the time of blood product transfusion and explicitly advised against repeat laboratory testing  before the procedure. The thresholds for PLT, INR, and TEG parameters were determined by a multidisciplinary group of physicians in hepatology, interventional radiology, and transfusion medicine. Multiple interdisciplinary meetings were performed to review existing relevant literature on the topic and develop the TEG-based protocol. These faculty meetings occurred from January to April 2019.In May 2019, we enacted a TEG-based transfusion protocol utilizing an evidence-based algorithm to guide blood product transfusion  included dissemination of the protocol among providers in charge of paracentesis and education on the interpretation of TEG results. We accomplished this by posting copies of the protocol in pertinent clinical working areas (including the dedicated area for outpatient paracentesis and the hepatology inpatient service), training nurses and advanced practice providers involved in the care of patients undergoing paracentesis, and frequently reminding protocol workflow during hepatology faculty meetings as well as in internal grand rounds or other educational efforts for trainees. As a key component of implementation, the authors gave special emphasis on the education of providers placing the orders for paracentesis so that they could identify when to request the TEG and the need for transfusion and on the providers performing the paracentesis so that they could confirm the transfusional intervention was properly executed before paracentesis. The senior author (Andres Duarte-Rojo) made himself available to any provider in need of advice, and our transfusion medicine expert (Darrell Triulz) was consulted when in doubt. In consideration of a suboptimal uptake of the TEG-based protocol during the initial phase of implementation, we eliminated subjects who underwent paracentesis during May and June of 2019. The TEG-based protocol was registered as a Quality Improvement Project at the corresponding UPMC website and approved by overseeing authorities.We collected clinical and demographic data, including age, sex, ethnicity, BMI, etiology, cirrhosis-related complications, and other medical comorbidities . All patients had pre-paracentesis hemoglobin, serum electrolytes, renal function, liver function panel, and coagulation studies, as well as post-paracentesis hemoglobin and serum creatinine. Procedural data were also collected, including paracentesis setting , ascites volume drained during paracentesis, and dose of albumin given after the procedure. Lastly, any preprocedural transfusion of PLT, plasma, and cryoprecipitate was also recorded. Charts were independently reviewed by 7 investigators . Direct costs of blood products transfused and the direct cost of TEG assay performance were obtained from 2021 internal operations cost data from the Division of Laboratory Medicine, Department of Pathology at the University of Pittsburgh. Costs were in US dollars.p < 0.05. All analyses were performed using the Stata 16.1 software . This study was approved by the University of Pittsburgh’s Institutional Review Board. The manuscript was reviewed and approved by all authors prior to submission.Demographics and clinical characteristics of the cohort were described using frequency and percentages for categorical variables, while continuous variables were listed as mean ± SD or median (25th–75th percentiles). Chi-square, Student’s t, or Wilcoxon tests were used for comparisons, as appropriate. Statistical significance was defined as http://links.lww.com/HC9/A599. With respect to paracentesis equipment, 184 (14%) paracenteses were performed for diagnostic purposes with a spinal needle, whereas 1097 (86%) were performed with a 5- or 6-French nonlocking catheter.We identified 483 patients with cirrhosis who underwent a total of 1281 paracenteses during the study period. The pre-TEG protocol cohort consisted of a total of 253 patients with cirrhosis who underwent 607 paracenteses from September 2017 to April 2019. The post-TEG protocol cohort consisted of a total of 230 patients with cirrhosis who underwent 674 paracenteses from July 2019 to September 2021  as did 1 patient after the TEG-based protocol cohort (0.4%). However, on further review of the post-TEG cohort patient’s course, it was determined that the episode of PPH was secondary to operator technique, causing an inadvertent arterial puncture based on direct visualization of extravasation from inferior epigastric artery seen on post-paracentesis abdominal imaging. Neither patient received pre-paracentesis blood product transfusions.The mean direct costs for these blood products, as well as the cost of performing TEG at our center during the study period, were obtained and compared based on product utilization rates between the 2 cohorts Table . The oveIn our study, the implementation of a TEG-guided transfusion protocol resulted in a significant reduction in preprocedural blood product utilization compared to prior utilization rates based solely on conventional parameters . Additionally, this decrease in prophylactic blood product transfusions was safely tolerated, with no increased incidence of PPH occurring in the post-TEG protocol cohort. The associated decrease in preprocedural blood transfusions following TEG-based transfusion protocol demonstrates strong potential for cost savings without compromising the quality of care. These findings substantiate the use of TEG as a safe and effective method for guiding prophylactic preprocedural blood product transfusions in patients with cirrhosis.16 Despite such recommendations, abnormal PLT and INR levels are still often cited by providers as informing preprocedural blood product utilization. Of note, the INR and PLT thresholds stated in our TEG protocol algorithm  were based on consensus from an institutional multidisciplinary protocol review process, and the thresholds are more abnormal than standard definitions of secondary coagulopathy and thrombocytopenia used by others, including professional societies.18 Despite using these higher thresholds of conventional markers of presumed increased procedural bleeding risk, we observed a marked decrease in prophylactic blood product use among the post-TEG cohort with no associated increase in procedural bleeding complications. This supports that TEG was useful in estimating the risk of PPH in patients with cirrhosis despite abnormal conventional parameters  and was a better tool to inform appropriate blood product utilization in this patient population. These positive outcomes should prompt consideration for revising the “standard” conventional parameters historically driving prophylactic preprocedural transfusion strategies.Accurately predicting periprocedural bleeding risk in patients with cirrhosis is challenging. Given the aforementioned limitations of INR and PLT to determine bleeding risk in these patients, current guidelines advise against prophylactic preprocedural blood product transfusions based on these conventional parameters.19 Our study demonstrates that such education can directly and safely reduce unnecessary prophylactic transfusions that can spare patients’ associated health risks and reduce health care spending.Furthermore, by virtue of using these higher thresholds to determine the need for TEG, it is imperative to note that TEG was required for only 104 of the entire 674 paracenteses (15%) performed in the post-TEG cohort. This suggests that educating providers on the interpretation of conventional lab parameters to determine periprocedural bleeding risk was a leading contributing factor to the observed decreased blood product utilization. The value of provider education on clinical determination of blood product utilization in patients with cirrhosis cannot be underestimated, especially when taking into account the increased morbidity and mortality posed by transfusion-associated complications  among patients with advanced liver disease.20 This highlights the additional clinical value of utilizing TEG to guide periprocedural prophylactic blood product use among patients with cirrhosis.Importantly, some patients with cirrhosis who are waitlisted for a liver transplant also require renal transplantation either simultaneously or after liver transplant. In such patients, it is critical to limit unnecessary blood transfusions to prevent allosensitization, which is associated with increased waitlist time, increased risk of graft rejection, and lower graft survival compared to nonsensitized patients.22 Along with this comprehensive assessment, TEG also provides several other advantages, including its ability to provide rapid, real-time results that are amenable to guide blood transfusion strategies in a preprocedural setting.23 In a systematic review and cost-effectiveness analysis of the use of TEG in patients deemed high clinical bleeding risk , Whiting et al demonstrated that TEG analysis resulted in less transfusion of blood products, reduced complications, decreased hospitalization costs, and a reduction in mortality.24 Our study similarly found that improved resource utilization produced noninferior clinical outcomes while achieving associated decreased costs of care, though formal survival and cost analyses were not performed.In contrast to conventional parameters of coagulopathy, viscoelastic testing such as TEG provides a more global assessment of hemostatic function, including clot formation, stability, fibrinogen levels, platelet function, and thrombin generation.It is worth noting that our study has some limitations. First, this is a single-center study, which can predispose to limited generalizability of our findings. However, our large total cohort size, coupled with the strong similarity of several clinical factors among the cohorts, provided adequate power to detect significant results between the 2 groups. Secondly, our designated follow-up time consisted of 48 hours after paracentesis and, therefore, could have theoretically limited detection of delayed PPH, which can occur several days after paracentesis. Thirdly, we did not assess the fibrinogen level, which can impact the TEG MA results that we used to determine the need for platelet transfusion. Cases receiving cryoprecipitates did so based on available fibrinogen levels, which were obtained at the discretion of the attending physician and not contemplated by our protocol. The multidisciplinary collaborative effort, including a thorough pertinent literature review, that was used to develop this protocol’s transfusion algorithm does lend itself to further protocol revision should future studies demonstrate the utility of pre-paracentesis fibrinogen levels or incorporation of κ time/α angle into decision-making to prevent PPH among patients with cirrhosis. Lastly, primarily due to some constraints in accessible cost data at the time of the study, our cost analysis was restricted to direct transfusion costs at our center to assess for cost differences between the pre-TEG and post-TEG cohorts. Formal cost-effective analysis, including sensitivity analysis, is needed to better determine the potential for cost savings with periprocedural prophylactic TEG-based transfusion protocol in this patient population.In conclusion, the implementation of a pre-paracentesis TEG-based transfusion protocol for patients with cirrhosis successfully resulted in decreased blood product use with no increased incidence of PPH. TEG-based protocols appear to better inform providers about appropriate pre-paracentesis blood product use and can contribute to higher-value care, though further studies, including formal cost-effectiveness analysis, are warranted.


Transcatheter aortic valve implantation (TAVR) is the first therapeutic option for elderly patients with severe symptomatic aortic stenosis, and indications are steadily expanding to younger patients and subjects with lower surgical risk and longer life expectancy. Commissural alignment between native and transcatheter valves facilitates coronary access after TAVR and is thus considered a procedural goal, allowing long-term management of coronary artery disease. Moreover, commissural alignment may potentially have a positive impact on transvalvular hemodynamic and valve durability. This review focus on technical hints to achieve commissural alignment and current evidence for different transcatheter aortic valves. Transcatheter aortic valve replacement (TAVR) has become the standard treatment for elderly patients with severe, symptomatic aortic stenosis (AS) across all surgical risk strata. More recently, the indications for TAVR have expanded to include a younger patient cohort with fewer comorbidities.Coronary artery disease (CAD) frequently occurs concomitantly with AS due to shared predisposing factors and pathophysiology . ApproxiRecent studies have reported an incidence of acute coronary syndrome (ACS) following TAVR in the order of 5% at a median follow-up of 1 year ,7 and 10The ease of coronary engagement after TAVR depends on an interplay of several factors related to the native anatomy of the patient, the transcatheter heart valve (THV) design, and implantation characteristics , neo-commissures may face coronary ostia and create a potential anatomical barrier to future engagement. Accordingly, improvement in THVs and delivery systems to help achieve commissural alignment are nowadays at the forefront of attempts to optimize long-term management of coronary artery disease after TAVR.n = 483; Evolut, n = 245; ACURATE neo, n = 100) were evaluated using pre-procedural multidetector row CT and post-implant co-planar fluoroscopy co-registration. The Evolut THV platform has one of the commissures orientated in the same direction as the C-paddle, which is at an angle of 90° to the “Hat” marker, Tang et al. evaluated the impact of the initial THV deployment orientation on neo-commissural alignment and overlap with coronary arteries . In the p < 0.05). Orientation of the flush port of the delivery system at three o’clock (pointing towards the left of the patient) on insertion into the right femoral artery achieved a desired “Hat” in 91.6% of cases. The corresponding orientation of the flush port for the ACURATE neo2 THV  is six o’clock, and for the Navitor THV  is 12 o’clock. Unlike the self-expandable platforms, however, the initial crimped orientation of the SAPIEN 3 THV, consisting of three fluorogenic commissural tabs with four struts in between, had no impact on the final valve orientation.The cohort with “Hat” on the outer-curve (OC) or center-front (CF) of the aortic annulus prior to THV deployment had improved commissural alignment and a lower incidence of coronary overlap compared those with “Hat” on the inner curve (IC) and center back (CB) , reflecting the diversity in patient anatomies . ConsequBieliauskas et al. recently proposed a fluoroscopy-based, patient- and device-specific TAVR implantation technique for several self-expanding THV platforms . In the After insertion of the system with the flush port at three o’clock for Evolut THV, six o’clock for ACURATE neo2 THV, and 12 o’clock for Navitor THV, a consistent implant sequence was followed. On reaching the level of the native annulus, the Evolut THV was slowly torqued until the “Hat” marker was in the CF position and the C-paddle was to the right of the screen in the cusp overlap view before THV deployment. Thereby, the THV commissures are aligned with the commissures of the native valve. For the ACURATE neo2 THV, the commissures are located at the same position as the posts, and the ‘free stent struts’ are visible on fluoroscopy. The commissure posts located mid-stent frame are less visible on the current generation Navitor THV. Post-TAVR CT assessment revealed that of 60 evenly distributed patients across these three platforms, optimal alignment (CMA < 15°) or mild commissural misalignment (CMA < 30°) was achieved in 53 patients (88%). No moderate or severe commissural misalignment was seen with the ACURATE Neo2 platform due to clearly visible landmarks for the commissures and easy torque of the delivery system.Kitamura et al. readapted the same technique on patients with failed surgical bioprosthetic valves undergoing valve-in-valve treatment with Evolut platforms . A preliDe Marco et al. used an alternative patient-specific algorithm to achieve commissural alignment with the ACURATE neo THV . The sexOther self-expandable THV platforms are available now, and similar workflows to achieve commissural alignment during implantation have been described. Hydra THV  has three wide, tentacle-shaped arches in the outflow tract, with three commissural posts rising up in between. Six radiopaque markers in two rows are located on the stent frame, to guide valve positioning. These markers highlight the center points of the three THV cusps. To achieve commissural alignment, two markers should be overlapped at the right of the screen in RCC-LCC cusp overlap view, isolating one marker on the left. A “near 1:2 pattern” has been shown to result in good CA and easy coronary engagement after valve positioning , while aTo date, there is no documented method on how to achieve commissural alignment, with the consequence that 33–43% of SAPIEN 3 THVs are deployed with moderate or severe misalignment .p < 0.001), with the non-coronary cusp being the most common dominant cusp . O. O22]. ONotably, in the study by Wang et al., the right–left cusp overlap view and coronary ostia overlap view differed by less than 10° of fluoroscopic angulation in three out of four cases, upon CT analysis of 200 tricuspid and 200 bicuspid aortic valves . Only 2.In specific cases with marked cusp asymmetry and/or coronary ostium eccentricity, a coronary ostia overlap view instead of a cusp overlap view could therefore be used during TAVR in order to position one of the neo-commissures centrally between the RCA and LCA . This stFacilitating future coronary access has highlighted the importance of commissural alignment during THV positioning Apart from the long-term management of coronary artery disease, the percutaneous treatment of younger patients raises concerns about valve durability, and strategies to optimize THV implantation, improve transcatheter valve function, prevent valve deterioration, and allow for THV-in-THV procedures are now paramount. The geometry of THVs after deployment plays a leading role in valve expansion and functionality. Whether the rotational orientation of a prosthetic device can impact the morphology and affect the physiology of THV leaflets is still a concern, with possible implications for valve durability. A rationale to investigate the effects of misalignment on THV hemodynamics was first provided by Bailey et al., who showed on a computational model that the rotational orientation of the device can affect neo-leaflet expansion . In theiFollowing this, clinical observational experiences provided controversial results on the possible correlation between commissural alignment, trans-valvular flow conditions and clinical outcomes.In 212 TAVR patients enrolled in the SAVORY and the RESOLVE registries, moderate or greater misalignment was shown to be an independent predictor of mild central aortic regurgitation at discharge for both self- and balloon-expandable THVs. Higher rates of central leaks for misaligned valves were confirmed at 3 month follow-up . The CMASubclinical leaflet thrombosis is a common finding in both surgical and transcatheter bioprosthetic aortic valves. Although it is uncertain whether this imaging phenomenon may lead to any clinical consequences, there are concerns that it may adversely affect valve durability and lead to thrombo-embolic events .The coagulative cascade can be triggered by low-flow conditions inside the neo-sinuses, and the morphological features and wash-out of these spaces can theoretically be affected by the rotational orientation of the device. A post hoc sub-analysis of the Low-Risk TAVR (LTR) Trial compared patients showing CT signs of subclinical leaflet thrombosis 30 days after TAVR to those who showed preserved leaflet morphology in order to identify possible predictors of this imaging phenomenon . Among cThese contradictory findings about a possible association between commissural alignment and leaflet thrombosis still require further investigation.Recently, the impact of commissural alignment on valve hemodynamic and related clinical outcomes has been retrospectively analyzed in a population of 324 consecutive patients treated with balloon-expandable THVs . No overAs TAVR is being offered to younger patients whose life expectancy may exceed THV durability, redo TAVR procedures are expected to become more common in the future. This possibility deserves consideration and should influence initial valve choice and deployment technique. In redo TAVI, the new valve displaces the degenerated leaflets of the first THV between two stent frame layers. This creates a “neo-skirt” that can reach sinotubular junction (STJ) level and result in sinus sequestration, with abrupt and life-threatening impairment of coronary flow. This risk is higher if the distance between the stent frame of the first valve and the STJ is short, especially in degenerated THVs with supra-annular leaflet design in which the neo-skirt may cover the SOV entirely . In redoIndications for TAVR have steadily expanded over the last decade, and nowadays include most elderly patients with severe AS across the entire spectrum of surgical risk. Percutaneous treatment of younger subjects with longer life expectancy poses new challenges to transcatheter therapy in which lifetime management is a predominant concern and optimizing clinical outcomes is mandatory. Optimization has been focused on several aspects of THV design and the implantation procedure. Commissural alignment has gained attention since observation experiences have shown easier coronary re-access and safe redo TAVR in cases of valve failure. A possible impact on valve durability and subclinical leaflet thrombosis needs further investigation.


Acute coronary syndromes due to atherosclerotic coronary artery disease are a leading cause of morbidity and mortality worldwide. Intraplaque hemorrhage (IPH), caused by disruption of Intraplaque leaky microvessels, is one of the major contributors of plaque progression, causing a sudden increase in plaque volume and eventually plaque destabilization. IPH and its healing processes are highly complex biological events that involve interactions between multiple types of cells in the plaque, including erythrocyte, macrophages, vascular endothelial cells and vascular smooth muscle cells. Recent investigations have unveiled detailed molecular mechanisms by which IPH leads the development of high-risk “vulnerable” plaque. Current advances in clinical diagnostic imaging modalities, such as magnetic resonance image and intra-coronary optical coherence tomography, increasingly allow us to identify IPH in vivo. To date, retrospective and prospective clinical trials have revealed the significance of IPH as detected by various imaging modalities as a reliable prognostic indicator of high-risk plaque. In this review article, we discuss recent advances in our understanding for the significance of IPH on the development of high-risk plaque from basic to clinical points of view. Atherothrombosis is a leading cause of morbidity and mortality worldwide . Three pIntraplaque hemorrhage (IPH) E is thouIn this review paper, we discuss the recent understanding of IPH, one of the key features of the unstable plaque phenotype, and its potential for future coronary event prediction from basic to clinical points of view.−/− and LDLR−/−) are usually applied as reliable in vivo models for human atherosclerosis. However, IPH, neovascularization and plaque rupture are not commonly detected in their atheromas. Thus, human pathology is still the major focus for research on IPH and neovascularization. In human pathologic sections, IPH can be generally detected by hematoxylin-eosin (H&E) or Masson trichrome stains, which are non-specific for hemorrhage detection. Additionally, further specific information can be obtained with special staining, such as Prussian blue (for iron deposition detection), and immunostaining with glycophorin A  [Since the 1930s, IPH has been described as one of the features of complex advanced atherosclerotic lesions ,13,14. Iembrane) . In a prembrane) . IPH incembrane) .Intraplaque vasa vasorum are reported to have incompetent barrier function of the endothelial layer. The disruption of endothelial integrity, which is generally maintained by intercellular junctions, results in the leakage of blood constituents from neovessels ,20. A prHemodynamic effects also contribute to the development of IPH. Prior clinical evidence supports the fact that arterial thrombotic occlusion tends to occur at or near the proximal region of the arterial stenosis . It is k2+) and ferric states (Fe3+) contributes to the formation of reactive oxygen species (ROS) through the Fenton reaction [Erythrocyte membranes contain a large amount of free cholesterol, and it is thought that the accumulation of erythrocyte membranes in atheromas due to IPH is a critical source of the lipid component of the necrotic core and the formation of cholesterol crystals composed of unesterified cholesterol. Free cholesterol retention in cells and tissues lead monohydrate crystal formation, which is originally derived from intra-cellular hydrolyzation of endocytosed cholesterol esters  or comesreaction . Local Rreaction ,49. Oxidreaction  and assireaction . TherefoIt is known that macrophages differentiated from peripheral blood monocytes are crucial contributors to the progression of advanced plaques . MonocytIn our current understanding, the differentiation of monocytes to macrophages is determined by the local environment and the availability of cytokines. Gordon et al. described the theory that microenvironments direct monocytes into functionally distinct cell types . AccordiRecent developments in single cell RNA-sequencing (sc-RNA seq) technology are unveiling further characteristics of macrophage heterogeneity in human atheromas. In the sc-RNA seq analysis of human atheroma in symptomatic and asymptomatic carotid artery disease, by Fernandez D. M. et al., six different subpopulations of macrophage in the carotid plaque were classified into six different subpopulations based on transcriptomic signature . These d+ macrophages and plaque vulnerability in their pathologic study that used 200 human carotid plaques removed from 197 patients by endarterectomy [+ macrophages in the atheroma was positively correlated with the atherosclerosis progression and the number of microvessels in the plaque ). Supernatants from M (Hb) increased EC capillary tube formation, vascular permeability, and the inflammatory response of the vascular endothelium [−/− and ApoE−/−CD163−/−) revealed a significantly lower atheroma burden, necrotic core size, lesion stenosis and micro angiogenesis in ApoE−/−CD163−/− as compared to ApoE−/− mice, implicating CD163 in atheroprogression. It was confirmed that the peritoneal macrophages from CD163−/− mice had significantly lower VEGF production when cultured under hemoglobin stimulation than those of wild-type mice [+ macrophages at the site of IPH contribute to the plaque development and destabilization by releasing VEGF. Local VEGF upregulation enhances vascular permeability and inflammatory cell infiltration into the plaque, thereby promoting further bleeding from immature neovasculature [In vitro experiments using human, peripheral blood mononuclear cells showed that VEGF production was enhanced from Hb:Hp-stimulated CD163othelium . These eype mice . CD163+ culature .Coronary artery calcification is known to be a manifestation of advanced atherosclerotic disease. The detailed characteristics of calcification are related to the specific phenotypes of atheroma ; i.e., d+ macrophages and Intraplaque vascular SMC [+ macrophages and calcification. In vitro experiments using vascular SMCs cultured with a supernatant of M (Hb) showed anti-calcific effect while arteries from ApoE−/−CD163−/− mice showed greater vascular calcification compared with that from ApoE−/− mice. M (Hb) supernatant-exposed SMCs showed activation of the NF-κB pathway. Blocking this signaling pathway attenuated the anti-calcific effect of M (Hb) on SMCs. M (Hb) altered calcification through NF-κB-induced transcription of hyaluronan synthase (HAS), an enzyme that catalyzes the formation of the extracellular matrix glycosaminoglycan and hyaluronan within SMCs. M (Hb) supernatants enhanced HAS production in SMCs, while knocking down HAS attenuated its anti-calcific effect. NF-κB blockades in ApoE−/− mice reduced hyaluronan and increased calcification in the plaque. In human arteries, hyaluronan and HAS were increased in areas of CD163+ macrophage presence. These findings show an important mechanism by which CD163+ macrophages (in the plaque) inhibit extensive calcification through NF-κB-induced HAS augmentation in SMC, promoting the high-risk vulnerable plaque [Recently, we further elucidated the contribution of IPH to the development of less-calcified vulnerable plaque phenotypes through interaction between CD163ular SMC . Patholoe plaque .Taken together, at the sites of IPH: (1) the accumulation of free cholesterol in the necrotic core due to erythrocyte membrane deposition A, (2) liThe pathological findings of IPH site within the plaque undergo constant evolution over time . In addition, since most IPH often occurs within or beside the necrotic core, necrotic tissue and lipid components might coexist with various degrees of mixture at IPH sites, affecting the prolonged local healing time sequence. It might be difficult to recognize IPH as a uniformly identifiable lesion by clinical diagnostic imaging modalities. In this regard, to date, magnetic resonance imaging (MRI) in carotid atherosclerosis is the most established diagnostic tools for the detection of IPH, which, based on iron detection with T1-weighted sequences, gives an Intraplaque hyperintense signal, whereas other components are detected as isointense or hypointense signals . Particup = 0.009) and lipid-rich necrotic core volume  was significantly greater in IPH (+) vs. in IPH (−) group [p = 0.006) and greater decreases in lumen area  in plaques with IPH during two-years follow up in patients on continued lipid-lower therapy [Several prospective clinical trials have shown that the presence of MRI-defined IPH in carotid atheroma is a robust predictor for future risk of ipsilateral ischemic stroke and/or transient ischemic attack (TIA) . Takaya −) group . Another therapy . These f therapy . In the  therapy . Evaluat therapy .Other modalities, such as contrast-enhance computational tomography (CT), have also been reported to be able to distinguish IPH from lipid-rich and fibrous parts in carotid atheromas ,78. SabaAs an alternative, a novel technique was developed to detect the co-existing neovascularization in high-risk plaque . Serial Because coronary arteries and their atheromas are quite smaller than carotid arteries, it is also more difficult to analyze these vessels due to their movement with the heartbeat. Thus, determination of IPH in coronary arteries using imaging tools is tough to accomplish. Nevertheless, recent advancements of coronary imaging tools have been allowing us to detect likely IPH lesions in coronary atheromas. Noguchi et al. prospectively investigated whether coronary hyperintensity atheromas (HIP), as observed by non-contrast T1-weighted turbo field echo sequence images on cardiac MRIs, were predictors of future coronary ischemic events in 568 patients . A patho2. Taken together, IPH is one of the robust characteristics related to the prognosis of unstable atheroma.It has recently been reported that intra-coronary plaque characterization by optical coherence tomography (OCT) is also useful to identify IPH lesions . The co-At present, one of the possible technologies for detecting in vivo biological IPH beyond plaque morphology is the near-infrared autofluorescence (NIRAF) imaging . Htun etRecent basic research has shown new insights on the manner in which IPH contributes to atheroma development and its vulnerability. Although lipid-lowering therapy has dramatically improved the prognosis of atherosclerotic disease, patients with LDL-C levels below the guideline-recommended target still have significant residual cardiovascular event rates . In this


Acute hepatitis A virus infection is routinely identified through a thorough patient history in conjunction with liver chemistries and viral serologies. The diagnosis has the potential to be delayed when the clinical picture is obscured with another, seemingly more urgent presenting pathology with overlapping features. Here, we describe the case of a young female who presented with acute calculous cholecystitis with concurrent acute hepatitis A virus infection. Acute hepatitis A virus (HAV) infection is a well-known, vaccine-preventable viral infection stemming from fecal-oral transmission. Since 2016, HAV outbreaks have been publicly reported in 37 states, totaling 44,915 cases, of which 61% required hospitalization and 423 led to death [A 36-year-old female with no significant past medical history presented with jaundice and mild, sporadic epigastric abdominal pain of three-day duration with associated dark urine and pale-colored stools. Physical examination revealed an afebrile, hemodynamically stable woman with point tenderness in the right upper abdominal quadrant. Workup was significant for a total bilirubin of 13.5 mg/dL (direct 9 mg/dL), alkaline phosphatase of 169 U/L, gamma-glutamyl transferase (GGT) of 136 U/L, alanine transaminase (ALT) of 586 U/L, and AST of 100 U/L. Ultrasound of the liver and gallbladder, as well as magnetic resonance cholangiopancreatography (MRCP), were significant for cholelithiasis with a 14.2 mm (1.42 cm) echogenic stone identified at the gallbladder neck with changes of acute cholecystitis  was not deemed necessary. The patient was taken directly for laparoscopic cholecystectomy with intraoperative cholangiography and liver biopsy on hospital day two. Intraoperative findings were consistent with acute cholecystitis without filling defects in the biliary tree.The patient’s bilirubin continued to rise post-cholecystectomy, peaking at 21.8 mg/dL. The hepatology team was then consulted and a viral hepatitis work-up was performed which returned positive for HAV immunoglobulin M (IgM). At the same time, her intra-operative liver biopsy also returned demonstrating acute viral hepatitis and frequent eosinophils in the muscular wall of the gallbladder Figures .A diagnosis of acute hepatitis A infection was confirmed, and the patient was treated symptomatically. Her AST and ALT gradually trended down over the subsequent few days. Her symptoms resolved, and she was discharged with plans for close clinic follow-up. Further history revealed that she had consumed frozen strawberries from a local grocery store, and recent news had implicated the spread of HAV via frozen strawberries from certain farms in the area.Despite lab work suggestive of hepatocellular injury and severe hyperbilirubinemia with jaundice, the diagnosis of HAV was delayed due to the concurrent presentation of acute cholecystitis and lack of a prodromal phase typical of HAV infection. Our case highlights the fallacy of an “anchoring bias,” wherein concurrent conditions are not considered because of complete reliance on the first piece of information received . AlthougThis case serves to illustrate the importance of further investigation of viral hepatitides in the setting of severe hyperbilirubinemia and jaundice, even if another related acute pathology is identified. In this particular case, the concurrent presentation of acute cholecystitis delayed the diagnosis of acute HAV infection likely secondary to symptom and laboratory result overlap as well as the urgency of the former diagnosis. A delay in proper diagnosis can be costly for patients who are elderly, immunocompromised, or those with pre-existing liver disease, therefore reiterating the importance of a timely and accurate diagnosis.This case also demonstrates the pitfalls of an anchoring bias and should alert the clinician to retain a high suspicion for further investigation if the clinical workup remains inconsistent with a previously diagnosed process. The degree of hyperbilirubinemia demonstrated in this case is not consistent with acute cholecystitis and in the absence of common bile duct blockage, should prompt the clinician to search for alternative explanations that may better explain the clinical scenario. Despite the absence of typical features of acute viral hepatitis, all cases with this degree of hyperbilirubinemia should undergo serological testing for acute viral hepatitides.


We present our experience of titanium-milled two-piece patient-specific implants (PSIs) for primary reconstructions of extensive orbital floor and medial wall fractures (EOFMFs) and evaluate their postoperative functional and aesthetic outcomes in relation to commercially available implants.We included all patients with primary reconstructions (< 22 days from injury) of EOFMFs treated in our department between January 2011 and October 2020. Extensive orbital floor and medial wall fracture was defined as involvement of orbital floor, medial wall and maxilloethmoidal junction; a fracture defect 5 mm or more; defect size more than a third of both inferior and medial walls; and Jaquiéry classification III or more. Patient characteristics, details of fracture defects and surgeries, postoperative outcomes and implant positions were retrospectively evaluated and compared between study groups.Nineteen patients were included: 5 with two-piece PSIs and 14 with commercial implants. Implant position was good in 4/5 patients with two-piece PSIs and 2/14 with commercial implants. Revision surgery, globe malposition (GMP) > 2 mm, significant diplopia and poor implant position were more frequent in patients with commercial implants than two-piece PSIs. None of the patients with a good overall implant position had any significant postoperative symptoms.Extensive orbital fracture reconstructions are somewhat rare, and surgical treatment is associated with a high rate of complications and postoperative symptoms. Titanium-milled two-piece PSIs are well suited for primary reconstructions of EOFMFs, as they lead to more precise reconstructions and fewer postoperative symptoms than commercially available implants. Orbital wall fractures may be blow-out fractures or fractures that involve the orbital rim in addition to the orbital walls . OrbitalSurgical reconstructions of EOFMFs are known to be challenging, especially if the maxilloethmoidal junction is involved as it functions as the anatomical cornerstone of the orbital cavity –7. AlthoThree-dimensional (3D) technology, including computer-aided design and manufacturing (CAD-CAM) techniques, are increasingly used in the treatment of orbital fractures 10). Orbital implants may be preoperatively prepared by bending them manually on a personalized skull-model or by making a virtual reconstruction by mirroring the unaffected contralateral side on the affected side as a reference . Orbital.As mentioned, the precision of the bony orbital cavity reconstruction is not the only factor that affects postoperative outcome. In EOFMFs, extensive surgical approaches and substantial surgical handling of the orbital soft tissue may cause marked iatrogenic soft tissue injury. However, if the surgeon attempts to avoid this soft tissue injury, a smaller implant may be chosen and, therefore, a part of the defect may be left uncovered. Both of these situations may lead to residual postoperative symptoms. Thus, two-piece PSIs may help the surgeon to perform a precise anatomical reconstruction, to reduce the iatrogenic orbital soft tissue injury  and to dThe aim of this retrospective study was to present our experience of titanium-milled two-piece PSIs for primary reconstructions of EOFMFs. In addition, we sought to evaluate their postoperative functional and aesthetic outcomes and postoperative complications compared to commercially available implants.We sought to compare the outcomes of titanium-milled two-piece PSIs and commercial implants in orbital fracture reconstructions. We retrospectively evaluated all patients with primary orbital fracture reconstruction treated in the Department of Oral and Maxillofacial Diseases, Helsinki University Hospital between January 2011 and October 2020.We included patients, who underwent a primary reconstruction of EOFMF with preoperative and postoperative high-resolution 16-slice computed tomography (CT) imaging into the study. Delay from injury to fracture reconstruction was required to be ≤ 21 days.Extensive orbital floor and medial wall fracture was defined according to five criteria: orbital floor and medial wall fracture; fracture of maxilloethmoidal junction; ≥ 5 mm fracture defect dislocation, defect size more than a third of both inferior and medial walls; and Jaquiéry classification ≥ III .We collected the following variables and compared them between the study groups: age, sex, delay from injury to surgery, facial fracture type , orbital fracture defect type  , occurreVariables regarding clinical postoperative outcomes included the following: requirement of orbital revision surgery; occurrence of GMP > 2 mm, eyelid malposition, any diplopia, diplopia that interfered with daily activities and diplopia that required strabismus evaluation by an ophthalmologist; requirement of additional procedures due to eyelid malposition.The authors evaluated the positions of the implants based on postoperative CT scan images. The overall anterior, medial and posterior positions of the plates were classified as good , acceptable  and poor  as previously described . In addiThe two-piece PSIs Fig.  were desData were analyzed with IBM SPSS Statistics for Mac . The results are shown as means (range) and numbers of named cases (percentages).Of the 266 orbital fracture patients requiring primary fracture reconstruction, 19 patients (7%) fulfilled the inclusion criteria and received reconstructions of unilateral EOFMFs (Fig. Surgical details are presented in Table The implants tended to be in a better position in patients with two-piece PSIs Table ; Fig. 2 Postoperative long-term follow-up data were available from 14 patients Table . OverallPatients with a poor implant position tended to have significant postoperative clinical symptoms; all of these patients had both significant GMP and some degree of diplopia Table .Table 4IIn this single-centre retrospective study, we presented a series of cases with reconstructions of EOFMFs. We found excellent postoperative outcomes in patients with two-piece PSIs; they had anatomically more precise reconstructions with less postoperative symptoms compared to patients with commercial implants despite their larger orbital defects. As EOFMFs are rare and may be challenging to reconstruct, our results suggest that two-piece PSIs can be recommended for their primary reconstructions to avoid further revision surgeries and other suboptimal outcomes.The aims of orbital reconstructions are to restore the anatomy of the orbital cavity , 15, retIn our series, most of the patients with commercial implants had malpositioned implants. In contrast, this was observed in only one patient with a two-piece PSI and was not clinically relevant. These findings reflect the challenges related to the process of fitting and aligning commercial implants, which may be time consuming and operator dependent. Importantly, these results also emphasize the advantages of CAD-CAM technology in reconstructing EOFMFs, as personalized CAD-CAM implants lead to more precise orbital volumetric reconstruction compared with traditional reconstruction methods , 19. AddEnophthalmos, restricted extraocular muscle motility and diplopia are a typical postoperative complication related to extensive orbital wall fractures , 20, 21.The delay between fracture diagnosis and primary surgery was longer in patients with PSIs than commercial implants. However, this difference was not clinically significant, as orbital fracture surgery can be safely performed within the first weeks of the trauma without leading to inferior postoperative outcomes .There is debate that the transconjunctival-retrocaruncular approach may cause postoperative complications, including persistent inferior oblique malfunction, inferior canalicular obstruction and scarring, which may result in diplopia . In our In our centre, the Helsinki protocol of two-piece PSIs is fairly simple and quick; the implant can be acquired within 1 day of order and thus does not cause a significant delay in surgery. Even if the manufacturing costs of PSIs for primary orbital reconstructions are evidently higher than using commercial implants, they lead to more precise implant positioning and better clinical outcomes. Thus, the costs of the overall treatment may eventually decrease and lead to less morbidity and stress to the patient. However, due to the importance of cost-effectiveness, the performance of primary orbital reconstruction with PSIs could be started in patients with large fractures, as they generally have poorer postoperative outcomes than patients with single-wall fractures.The correct placement of PSIs was easily evaluated without intraoperative navigation by the good fitting of the PSI over the fracture defect and the proper fixation between the two parts of the implant. Thus, two-piece PSIs may at least partially replace intraoperative navigation and imaging, which were not available in the present study. Despite the screwless fixation of two-piece PSIs, no failures of the attachment between the implant parts occurred.The greatest limitation of this study was the low number of patients, which precludes definite conclusions. Additionally, the retrospective and non-comparative study design and the limited rate of actualized clinical follow ups were also weaknesses. Moreover, due to the low number of patients and the variety of procedures performed during the primary surgery of the fracture reconstruction, this study did not assess the effect of PSIs on surgery duration.Titanium-milled two-piece PSIs are well suited for primary reconstructions of EOFMFs. These patients had fewer postoperative clinical symptoms than patients with commercial implants. This was possibly due to the less invasive surgical approaches, reduced iatrogenic soft tissue injury and greater precision of reconstruction. Therefore, we recommend the described two-piece PSI method for these most challenging primary orbital fracture reconstructions. However, to achieve even better postoperative outcomes for all orbital fracture patients, future research should focus on the role of damage to intraorbital soft tissues , 28 and


VG) in fitness related traits is often unexpectedly high, evoking the question how VG can be maintained in the face of selection. Sexually antagonistic (SA) selection favouring alternative alleles in the sexes is common and predicted to maintain VG, while directional selection should erode it. Both SA and sex-limited directional selection can lead to sex-specific adaptations but how each affect VG when sexual dimorphism evolves remain experimentally untested. Using replicated artificial selection on the seed beetle Callosobruchus maculatus body size we recently demonstrated an increase in size dimorphism under SA and male-limited (ML) selection by 50% and 32%, respectively. Here we test their consequences on genetic variation. We show that SA selection maintained significantly more ancestral, autosomal additive genetic variance than ML selection, while both eroded sex-linked additive variation equally. Ancestral female-specific dominance variance was completely lost under ML, while SA selection consistently sustained it. Further, both forms of selection preserved a high genetic correlation between the sexes . These results demonstrate the potential for sexual antagonism to maintain more genetic variance while fuelling sex-specific adaptation in a short evolutionary time scale, and are in line with predicted importance of sex-specific dominance reducing sexual conflict over alternative alleles.Genetic variance ( A positive rm,f sets the stage for intralocus sexual conflict when the sexes have different fitness optima [rm,f has been considered as a potential prerequisite to the evolution of sexual dimorphism [rm,f when compared across different traits [rm,f during the evolution of sexual dimorphism is not well understood. Studies comparing the rm,f across different types of traits [rm,f is estimated) are lacking.How selection affects genetic variance across the sexes depends on the degree to which shared genetic variation affects joint phenotypic variation in the two sexes, which is captured by the intersexual genetic correlation . se e.g. –43) show showrm,frm,f, are affected by evolution of sexual body size dimorphism (SSD) by artificial selection in a seed beetle Callosobruchus maculatus, and specifically test whether SA selection can maintain genetic variation at a higher level compared to sex-limited directional selection. Body size is commonly sexually dimorphic, and especially interesting trait given its strong association with life-history variation, ecological adaptation and speciation rates across taxa [C. maculatus population used for this study [C. maculatus body size  in a wild-type ancestral population and experimentally demonstrated that sexual size dimorphism increased under both SA selection  and male-limited (ML) selection , by 50% and 32%, respectively. These selection forms affected male size similarly ), while the SA selection prevented a correlational change in female size seen under ML selection, resulting in the greater increase in dimorphism under SA selection. We showed that the autosomal additive genetic variance was highly correlated between the sexes in the ancestral population . Nonetheless, predicting broadly, sex-limited directional selection is expected to erode additive genetic variance more quickly than SA selection. This is because sex-limited selection can cause an unhindered correlated response in the opposite sex , drivingPredicting the consequences of selection on the detected female-limited dominance variance requires also considering its effects on the opposite sex. Male limited-selection is completely blind to the effects of female dominance variance. If, on the one hand, the loci underlying female dominance variation have additive effects on males, ML selection is expected to push allele frequencies toward extreme values or fixation. If, on the other hand, the underlying loci have no effect on the male phenotype, selection on males only will leave such loci susceptible to drift. Consequently, female-specific dominance variance is expected to be reduced or lost under ML selection. By contrast, because heterozygosity is necessary for dominance effects to manifest themselves, and if sexual dimorphism is facilitated by the detected female-specific dominance, then SA selection should favour the expression of dominance effects by driving underlying allele frequencies to intermediate frequencies, where dominance variance is highest . If the rm,f is necessarily predicted under either selection regime, if SA maintains and ML selection reduces the VA equally in the sexes, but both forms of selection are expected to erode the sex-linked additive genetic variances. We test these predictions by comparing the variances between the replicated SA and ML lines, to the ancestral levels, as well as to the effects produced by genetic drift alone.Thus, unlike sex-limited directional selection, SA selection for increased sexual dimorphism could maintain or even increase dominance variance, in addition to maintaining the additive genetic variance. No change in the . 2 (a)b) . By contrast, the sex-linked additive variance components observed in males of the ancestral population were not maintained: Y-linked variance , and male X-linked genetic variance , male-limited (ML) or random selection (RS) . First, we found that a significant amount of autosomal additive genetic variance remained in both sexes after selection (ody size  was signVG) in the lines, SA selection preserved significantly more genetic variance across the sexes and replicate lines than ML selection . The pattern holds true whether analysing the replicate lines together or separately  or when analysed separately for each sex, SA selection maintained significantly more genetic variance in both females  and males  relative to ML selection. When partitioning VG to its additive and dominance components we also found that, in line with our main prediction, SA selection maintained significantly more additive genetic variance in both sexes when compared to ML selection , for both autosomal additive variance and female dominance variance . Comparisons of genetic variances between the SA and ML regimes to each random selection replicate are provided in the electronic supplementary material.Standing genetic variance estimates after 10 generations of selection were more consistent across the experimental replicates of the sex-specific selection regimes than under random selection, as we observed significant differences only between the two replicate lines of the random selection regime (RS (b)F1,5 = 0.5492, p = 0.49; In our previous study  we showeb and MLb .The intersexual genetic correlation is often calculated from quantitative genetic analysis without sex-linked and dominance variance partitioning which can bias the genetic correlation estimate ,50,51 , but can do so in the presence of large effect loci. Under selection, the stochastic effect of drift on large effect loci is likely secondary to the effect of selection itself. However, given both the persistence of periment , our resVME) (see methods). Regardless of sex, including selection lines where we found no dominance variance, VME was found to be zero (posterior mode = 0) in all cases. More importantly, in the ancestral population – where dominance estimates were not confounded with VME – we could not detect any maternal effects distinguishable from zero in either sex. In order for confounding effects of VME to explain our result of maintained female dominance, maternal effects specific to females would need to have arisen in the SA lines. We therefore believe that a more parsimonious explanation is that SA lines simply maintained the ancestral dominance variance and VME remained indistinguishable from zero.Maintenance of genetic variation in SA loci is explicitly predicted under sex-specific dominance ,36. In lSex-specific dominance variance both on the X  and on tThe increase in sexual size dimorphism under male-limited selection shows, however, that female-specific dominance variance is not necessary for dimorphism to evolve. The response of dimorphism to male-limited selection can be explained by the sex-linked variance in males, particularly on the Y chromosome detected in the ancestral population, which was further demonstrated with genetic crosses . The Y cG or VA, suggesting that SA selection also maintained more sex-limited genetic variance compared to ML. This indicates linkage of sex-shared and sex-specific genetic variances during the artificial selection. In principle, sex-shared and sex-specific genetic variances could be connected through pleiotropy, e.g. if a growth regulating locus affects both sex-shared and sex-specific tissues and thus contributes to sex-shared as well as to sex-specific variance. Alternatively, proximity of loci contributing to each type of variance could cause linkage. Whether sex-specific variances maintained due to linkage would persist over longer evolutionary times scales under SA selection depends on their underlying cause, but if caused by selection, linkage disequilibrium is expected to eventually break down over extended periods of time.Under the assumption that loci harbouring sex-shared and sex-specific variance are not linked, SA selection should specifically maintain sex-shared variance. The differences between SA and ML regimes should therefore be most pronounced when comparing the sex-shared variances (i.e. the between sex covariance). Albeit consistent in this direction, the difference in covariance we detected is not as large as seen in VThe amount of shared genetic variance remained high during 10 generations of selection and we could not detect a significant reduction in any of the selection lines compared with the ancestral population . Nor could we find any association between and the degree of change in sexual size dimorphism . This furelation . This isThe idea that selection for increased sexual dimorphism has the potential to maintain and even increase the d before ,32,33. Bc traits . The rm,pression . The patpression ,33, and ss genes  or highess genes ,58 that ss genes . These iss genes –61, whic. 4Our results experimentally show that sexually antagonistic selection can maintain autosomal additive genetic variance longer than sex-limited directional selection, which more easily erodes variation. The shared genome is a prerequisite for genetic variation to be maintained under SA selection, but also the reason why genetic variation is lost in both sexes due to directional selection even if only one sex is under selection. Our results additionally shed new light on the historic debate over the role of dominance variation evolution , by show. 5 (a)Callosobruchus maculatus is a highly amenable study organism to use in tests of the quantitative genetic underpinnings of sex-specific phenotypic evolution, as it shows heritable variation in the degree of sexual size dimorphism. As a pest of legume seed pods, female C. maculatus oviposit directly onto seeds in which the larvae conclude their development and emerge as mature adults after 21–25 days under benign conditions . C. macC. maculatus population used to establish the selection lines for this study was created by mixing 41 isofemales lines established from wild-collected beetles. The isofemale lines were founded by randomly pairing virgin beetles collected near Lome, Togo in 2010. More detailed information about the founding populations is presented in [The ented in . (b)In this study, we experimentally tested how two different forms of sex-specific selection, sexually antagonistic (SA) selection for larger females and small males  and sex-limited selection for small males (ML), as well as drift due to random selection, affected different genetic variance components associated with body size evolution during 10 generations of artificial selection. To do this, we compared the lines after selection to each other, to random selection, and to the ancestral population prior to the selection period. For this, we capitalized on data on the ancestral population presented in , where w (i)C. maculatus population before selection  using a pedigree design adapted from [Here we provide an overview of the quantitative genetic and artificial selection methods applied to the ancestral population. Further details can be found in . An exteted from  specific (ii)To reach comparable sample sizes to the quantitative genetic pedigree analyses conducted for ancestral generation we split the line replicates into two blocks. The present quantitative genetics experiments for all three selection regimes of replicate A were started in parallel after one generation of panmictic mating after selection. One generation later (i.e. after two generations of panmictic mating), the breeding design for the selection regimes in replicate B were started as well. We performed the quantitative genetics experiment for each selection regime and replicate as described below.V. radiata beans for 24 h of oviposition. Both crossing and oviposition were done under benign conditions in a climate chamber.Similar to the quantitative genetics experiment using in the ancestral population, we used a pedigree design adapted from  spanningC. maculatus (GGGP) from the panmictic population were transferred to a new set of V. radiata beans for oviposition. As in the ancestral population, adult beetles were removed 48 h later to unify the age of the following great-grandparental generation (GGP) and to control and equalize density across all regimes. GGP individuals were collected as virgins and randomly crossed to create 120 GP families. Note that beyond controlling for age and density, the panmictic mating as well as the random paring of GGGP and GGP generations serves two crucial purposes in our experiment: (i) minimizing potential relatedness structure which would violate assumptions of the animal model and (ii) removing potential build-up linkage disequilibrium that can bias additive genetic variance to be overestimated [To initiate the quantitative genetic experiments, randomly collected adult stimated .GP generation: From each GP family we selected one individual at random but balanced for sex, yielding 120 unrelated beetles, 60 females and 60 males, respectively. Males and females were randomly crossed to give rise to 60 P families.P generation: For each parental family we randomly selected 1 male and 4 females. Each male was sequentially crossed to 4 randomly selected females of different parental families, giving rise to 4 half-sib families.In total we measured approximately 2000 beetles in each regime and replicate . (c)a priori. The R code of our models is provided in the SI. In addition to the Bayesian framework we also used a likelihood based method to verify our findings with a different method and explore model comparison using the software ASReml R 4.0. All calculations of associated statistics such as sex specific heritabilities and To detect potential changes in genetic variances due to the response to artificial selection we analysed the pedigrees of each artificial selection and drift line with the animal model (mixed effect model) in the software R, v. 3.4.0 . We usedge NADIV . In the We created posterior differences between marginal posterior distributions of the replicate lines to compare the genetic variance component estimates against the ancestral population, as well as within and across selection regimes. Additionally, we compared the male-female covariance estimates between the replicates of the SA and ML regimes. The posterior difference itself is a posterior distribution and statistical inference can be drawn similarly as for all posterior distributions (i.e. primarily via 95% CI). After verifying that there were no significant differences between the replicate lines within the SA or the ML selection regimes, we estimated the genetic variance components for each selection regime (i.e. including both A & B replicate lines in the same model) to compare the effects of SA versus ML selection on genetic variance by creating a posterior difference distribution between the regimes. Additionally, we also present individually the posterior difference distribution comparison across all SA and ML replicate lines to demonstrate the consistency of our findings .