The difficulties and challenges of applying the HLH-2004 diagnostic criteria to early identification and diagnosis of haemophagocytic lymphohistiocytosis have been fully addressed in previous studies. However, the distribution of the diagnostic time lag of haemophagocytic lymphohistiocytosis and related patient characteristics remain unclear. This study investigated the time lags between symptom onset and diagnosis and between hospital admission and diagnosis among pediatric patients with haemophagocytic lymphohistiocytosis, and identified factors that associated with a shorter or longer diagnostic time lag. The cohort of patients with haemophagocytic lymphohistiocytosis was drawn from a tertiary children's hospital and consisted of 122 pediatric patients. The distributions of symptom-to-diagnosis and admission-to-diagnosis time lags were assessed. Clinical characteristics within 48 h of admission and the fulfillment of HLH-2004 diagnostic criteria were compared among admission-to-diagnosis time lag categories. Logistic regression analyses were conducted to identify factors associated with an admission-to-diagnosis time lag >3 days. The median interval from first symptom onset to HLH diagnosis was 12 days (range 4–71 days) and the median interval from hospital admission to HLH diagnosis was 2 days (range 0–23 days). The following factors were negatively associated with admission-to-diagnosis > 3 days: Epstein–Barr virus infection; admission through pediatric intensive care unit; diagnosis established without NK-cell activity and soluble CD25 tests; the performance of all readily available diagnostic tests for HLH (within 48 and 72 h); concurrent fever, splenomegaly, and cytopenias within 48 h; hemophagocytosis, hypertriglyceridemia and/or hypofibrinogenemia within 48 h; and elevated ferritin, total bilirubin, alanine aminotransferase, and prothrombin time within 48 h. Our findings suggest that performance of adequate diagnostic tests for HLH is essential for early diagnosis of HLH. Once suspected, immediate and adequate diagnostic tests for HLH should be arranged for PICU patients. Improvements in diagnostic procedures and monitoring plans are needed to promote early diagnosis of HLH. Haemophagocytic lymphohistiocytosis (HLH) is a disorder characterized by extreme immune activation, which results in hypercytokinaemia and immune-mediated injuries to multiple organ systems –3. HLH iHowever, initiating timely HLH treatment is hindered by the challenge of rapidly establishing the diagnosis. Given the non-specific clinical presentation of HLH and the overlap between its symptoms and those of other inflammatory diseases, such as sepsis, the diagnosis of HLH is often delayed , 14. MorAnalysis of the literature concerning delayed diagnosis of HLH indicates that both the scope of the problem and the characteristics of affected patients are unclear , 12, 21.This was a retrospective cohort study that included pediatric patients (0 to 18 years of age) discharged  from Hunan Children's Hospital in China with a diagnosis of HLH between June 2015 and October 2018. The chart review was conducted between May 2019 and November 2020. The survival status at day 30 after hospital admission was extracted from the medical record or followed up by a phone call. The exclusion criteria included patients diagnosed with HLH prior to the indicated hospital admission date; patients with essential diagnostic data missing; or patients > 18 years of age at the time of HLH diagnosis.The study protocol was reviewed and approved by the Medical Ethics Committee of the Hunan Children's Hospital (HCHLL-2019-40) and have been performed in accordance with the ethical standards as laid down in the 1964 Declaration of Helsinki and its later amendments or comparable ethical standards. The requirement for written informed consent was waived by the Medical Ethics Committee of the Hunan Children's Hospital.HLH was diagnosed according to the HLH-2004 criteria . AccordiIn the study center, all hospitalized patients underwent assessments of sustained fever length, splenomegaly, and routine blood analysis on hospital admission. Triglycerides and fibrinogen levels were also routinely checked on admission in most patients, but not all patients, according to the clinical presentation and suspected diseases. Ferritin and haemophagocytosis assessments were performed if HLH or other related diseases were suspected. The HLH diagnostic procedure in the study center was that if a patient met more than three HLH criteria in routine assessments, diagnostic tests were performed to determine HLH susceptibility. Initial evaluation typically took up to 48 h and most amended tests results  would be received within 72 h of admission. During the study period, NK-cell activity and sCD25 tests were not routine and were performed only if a patient demonstrated robust signs of HLH (or other relevant diseases), and results typically were not received within 72 h. Genetic test was performed in only a few patients due to high cost and long result turnaround time. Patients who have suspected HLH but do not fulfill the diagnostic criteria would be further monitored by repeating the diagnostic tests for HLH, however the test interval was determined based on the clinical presentation and was not standardized in the study center.The main study variable was the admission-to-diagnosis time lags. The admission-to-diagnosis time lag was defined as the interval (in days) between hospital admission and the day of confirmed HLH diagnosis. Because the diagnostic procedure for initial evaluation and amended tests for HLH could require up to 3 days, patients were categorized into two admission-to-diagnosis time lag groups: ≤ 3 days and > 3 days. Presumably, following the normal diagnosis procedure and using current diagnostic criteria, most patients with suspected HLH should have received a diagnosis in ≤3 days of hospitalization. Patients diagnosed after 3 days were likely to have progressed from subclinical HLH to HLH or had a delayed diagnosis. The patients' characteristics according to symptom-to-diagnosis time lag categories were also investigated. The symptom-to-diagnosis time lag was defined as the interval (in days) from the reported first symptom onset to the day of confirmed HLH diagnosis. Patients were categorized into two groups based on the median value of symptom-to-diagnosis time lag: ≤12 days and > 12 days.Because fever, splenomegaly, and cytopenias were routinely checked in general clinical practice and their fulfillment would raise susceptibility to HLH, we investigated whether the concurrent fulfillment of these three items was associated with a shorter diagnostic time lag. Tests of NK-cell activity and sCD25 are not immediately available in many clinical settings, which may limit the application of the HLH-2004 criteria. To determine how these two criteria affected the diagnostic procedure, we investigated the numbers of patients who could establish an HLH diagnosis without NK-cell activity and sCD25 tests, then analyzed their associations with diagnostic time lags. To investigate the performance of early diagnostic procedures among patients according to diagnostic time lag categories, we calculated the proportions of patients who received all readily available tests for HLH  within 48 and 72 h of hospital admission.Other investigated variables were: general characteristics, including age, sex, hospital admission department; survival status at day 30 after hospital admission; underlying diseases/potential triggers of HLH, including primary HLH, autoimmune disorders, malignancy, sepsis [diagnosed as described in ], infectConcomitant diagnosis, symptoms, and laboratory tests within 48 of admission were investigated, including all HLH-2004 diagnostic items except for sCD25 and genetic tests (none of the results was reported within 48 h), diagnoses, and other common lab tests like total bilirubin (TBil), alanine aminotransferase (ALT), prothrombin time (PT), activated partial thromboplastin time (APTT), and INR.Categorical variables were presented as absolute values and percentages. Continuous variables are presented as mean (standard deviation), or median, range and quartiles (Q1 and Q3), as appropriate. Between-group comparisons for categorical variables were conducted using chi-squared test or Fisher's exact test, as appropriate. Between-group comparisons for continuous variables were conducted by Wilcoxon rank sum test. Logistic regression analysis were conducted to assess the factors associated with an admission-to-diagnosis time lag >3 days. The correlation between the symptom-to-diagnosis time lag and the admission-to-diagnosis time lag was tested by Spearman correlation analysis. All tests were set two-tailed with a type 1 error rate fixed at 5%. Missing data was not imputed. All statistical analyses were performed using SAS 9.3 .Additional tests were conducted to explore the diagnostic characteristic according to factors which were found to be associated with a shorter or longer diagnostic time lag. Two sensitivity analyses were conducted to determine whether the results of between-group comparisons for 30-day OS rates would change if different cut-off points were used for the time lag categorization.p < 0.0001; also see Between June 2015 and October 2018, 145 patients were discharged with a diagnosis of HLH. Of these, 23 patients were excluded from this study, either because they had been diagnosed with HLH prior to the indicated hospital admission date or because essential information was missing, leaving 122 patients for the analysis . Four pap < 0.0001). Three patients were lost to follow-up at day 30. The 30-day OS rate for the study cohort was 68.9%. The differences in 30-day OS rates among time lag categories were not statistically significant (p > 0.05).p = 0.0001). Differences in other underlying diseases and concomitant diagnosis among time lag groups were not statistically significant. Regarding the HLH-related treatment, one between-group difference was observed for the usage of dexamethasone: more patients in the admission-to-diagnosis time lag >3 days group were treated with dexamethasone before a diagnosis of HLH was established .In this HLH cohort, nine patients (7.4%) were diagnosed with primary HLH; four patients (3.3%) had autoimmune disorders; six patients had malignancy (4.9%); 76 patients (62.3%) had concomitant sepsis; 113 patients (92.6%) had infection; and 83 patients (68.0%) had acute or chronic EBV infection. The EBV infection rate was significantly lower in the >3 days admission-to-diagnosis time lag group (45%) than in the ≤3 days group . The proportion of patients who met all three regularly checked criteria  did not significantly differ among time lag categories (p > 0.05). More patients in the ≤3 days admission-to-diagnosis time group could have an HLH diagnosis established without NK-cell activity and sCD25 tests, compared with the corresponding number of patients in the >3 days group .p-values <0.05). Patients in the ≤ 3 days group also fulfilled more HLH diagnostic items  within the first 48 h of hospital admission, compared with patients in the < 3 days group . The rates of performing all readily available HLH tests within 48 and 72 h were significantly higher in the admission-to-diagnosis ≤ 3 days group (p < 0.05). The results of between-group comparisons for concomitant diagnoses within 48 h are shown in p-values <0.05). Several symptoms and laboratory findings within 48 h of hospital admission were associated with an earlier diagnosis, including fever, splenomegaly, and cytopenias, hypertriglyceridemia and/or hypofibrinogenemia, and haemophagocytosis, as well as elevated ferritin, ALT, TBil, APTT, PT, and INR. Diagnosis of sepsis within 48 h was also negatively associated with admission-to-diagnosis > 3 days . Performance of HLH diagnostic tests within 48 and 72 h of hospital admission was significantly associated with a lower risk of admission-to-diagnosis > 3 days .p < 0.05, p = 0.0493) and had a lower 30-day OS rate . Patients with EBV infection had shorter symptom-to-diagnosis time lags and admission-to-diagnosis time lags (p = 0.0022) and had a better 30-day OS rate .Because admission through PICU and EBV infection were negatively associated with an admission-to-diagnosis time lag > 3 days, we compared the diagnostic characteristics between PICU-admitted and non-PICU-admitted patients , and betime lags . They alp > 0.05).In this HLH cohort, the median lengths between first symptom onset to HLH diagnosis was 12 days (range 4–71 days); and the median lengths between hospital admission to HLH diagnosis was 2 days (range 0–23 days), and 67.2% patients were diagnosed within 3 days of admission. Age < 1 year was associated with a higher risk of admission-to-diagnosis time lag > 3 days. The following factors were negatively associated with admission-to-diagnosis > 3 days: Epstein–Barr virus infection; admission through pediatric intensive care unit; diagnosis established without NK-cell activity and soluble CD25 tests; the performance of all readily available diagnostic tests for HLH (within 48 and 72 h); concurrent fever, splenomegaly, and cytopenias within 48 h; hemophagocytosis, hypertriglyceridemia and/or hypofibrinogenemia within 48 h; and elevated ferritin, total bilirubin, alanine aminotransferase, and prothrombin time within 48 h.Our findings show that the OS rates did not differ significantly among time lag categories, but these results do not imply that early diagnosis will not improve survival. Importantly, although treatment could affect OS, our study did not control for treatment, which interferes with comparison of OS rates among groups. For example, more patients in the admission-to-diagnosis > 3 days group received dexamethasone before confirmed diagnosis of HLH. Furthermore, patients within the different time lag groups had distinct baseline characteristics. Our data showed that patients diagnosed earlier (symptom-to-diagnosis ≤ 12 days) had a shorter symptom-to-admission interval, implying that the disease progressed quickly in at least some of the rapidly diagnosed patients. Notably, 50% of the patients diagnosed ≤ 3 days were admitted to the hospital directly to the PICU, but only 27.5% of the patients diagnosed >3 days were admitted directly through the PICU. Our previous study showed that PICU admission was a risk factor for early death in patients with HLH . AdditioAge <1 year was associated with a longer admission-to-diagnosis time lag. This association was less likely to be mediated by the difference in diagnostic procedures among different age groups, as comparisons of the days between first symptom to hospital admission, fulfillment of three regularly checked criteria, and the rates of early diagnostic tests within 48 and 72 h between < 1 year and ≥ 1 year age groups showed non-significant results. A possible explanation is that the disease progression pattern among different age groups was different and could have affected the HLH diagnostic time lag. The generalizability and the cause of this association worth further investigation, which could guide the improvement of diagnostic and management procedures among different age groups.EBV is the most consistently reported viral infection associated with HLH, especially in Asian populations , 34–36. p < 0.0001). Consequently, patients with more HLH-like features underwent diagnostic/monitoring tests sooner than did patients with fewer HLH-like features, in agreement with our findings that the performance of all early tests for HLH (within 48 and 72 h) was associated with earlier diagnosis. A prolonged time lag between hospital admission and diagnosis may result from insufficient testing, but also could be caused by disease progression in patients who initially present with mild symptoms that later progress to obvious HLH during hospitalization (The most frequently met HLH-2004 criteria were cytopenias (100%), hypertriglyceridemia and/or hypofibrinogenemia (100%), splenomegaly (91.8%), and fever (91.0%). These findings were similar to those in a previous study . Consistlization . In the Both the laboratory results turnaround time and availability of diagnostic tests can affect the efficiency of diagnostic procedure. The current diagnose procedure in our study center took up to 3 days . Under this procedure, only 15.6% patients received HLH diagnostic test results within 48 h, and this rate increased to 63.1% within 72 h. Therefore, to establish a rapid diagnostic procedure for HLH, improvement in laboratory results turnaround time is needed in our center.A difficulty in applying the current diagnostic criteria is that tests for NK-cell activity and sCD25 are typically not available in low-resource settings . In someThe difficulties in applying the current diagnostic criteria for the early identification and diagnosis of HLH have been fully addressed in previous studies and review articles , 18, 21.The main limitation of our study was that it was a retrospective study conducted at a single center. Because of the retrospective design, the monitoring plan were not standardized in this study. The ability to rapidly diagnose HLH requires experienced medical staffs and sufficient resources, including the ability to conduct genetic, NK-cell activity, and sCD25 testing. The distribution of the diagnostic time and the influencing factors may vary depending on the study setting. However, this is the first study to examine the distribution of HLH diagnostic times in HLH and the associated patient characteristics. Therefore, our results provide an important reference for future studies. Hunan Children's Hospital is one of the largest tertiary children's hospitals in central China and has treated hundreds of pediatric HLH patients. Thus, our findings are likely to be generalisable, especially for regions with a comparable setting and similar medical resources. The second limitation also derived from the retrospective design: failure to conduct all necessary diagnostic tests, especially NK-cell activity, sCD25, and genetic tests, may have led to some missed HLH diagnoses. Lachmann et al. retrospectively collected the medical records of adult intensive-care-unit patients with a serum ferritin level of ≥ 500 μg/L and available data for at least four HLH-2004 criteria, then retrospectively diagnosed those patients with HLH ; they foTo conclude, performance of adequate diagnostic tests is essential for early diagnosis of HLH. A shorter diagnostic time lag could be a sign of rapid deterioration. Once suspected, immediate and adequate diagnostic tests for HLH should be arranged for PICU patients. Improvements in diagnostic procedures and monitoring plans are needed to promote early diagnosis of HLH.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by Medical Ethics Committee of the Hunan Children's Hospital. Written informed consent from the participants' legal guardian/next of kin was not required to participate in this study in accordance with the national legislation and the institutional requirements.ZX, XLu, XLi, and HY designed this study. ZX, XLi, and HY wrote the manuscript. All authors participated in the data analysis, interpretation, and agree to be accountable for the content of the work.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Antenatal anxiety has been linked to adverse obstetric outcomes, including miscarriage and preterm birth. However, most studies investigating anxiety during pregnancy, particularly during the COVID-19 pandemic, have focused on symptoms during the second and third trimester. This study aims to describe the prevalence of anxiety symptoms early in pregnancy and identify predictors of early pregnancy anxiety during the COVID-19 pandemic.We assessed baseline moderate-to-severe anxiety symptoms after enrollment in the UCSF ASPIRE (Assessing the Safety of Pregnancy in the Coronavirus Pandemic) Prospective Cohort from May 2020 through February 2021. Pregnant persons < 10 weeks’ gestation completed questions regarding sociodemographic characteristics, obstetric/medical history, and pandemic-related experiences. Univariate and multivariate hierarchical logistic regression analyses determined predictors of moderate or severe anxiety symptoms . All analyses performed with Statistical Analysis Software (SAS®) version 9.4.p < 0.0001), a pre-existing history of anxiety (p < 0.0001), and a history of prior miscarriage (p = 0.0143) were strong predictors of moderate-to-severe anxiety symptoms. Conversely, having received care at a fertility center was protective . COVID-19 related stressors including job loss, reduced work hours during the pandemic, inability to pay rent, very or extreme worry about COVID-19, and perceived stress were strongly predictive of anxiety in pregnancy (p < 0.0001). In the hierarchical logistic regression model, pre-existing history of anxiety remained associated with anxiety during pregnancy, while the significance of the effect of education was attenuated.A total of 4,303 persons completed the questionnaire. The mean age of this nationwide sample was 33 years of age and 25.7% of participants received care through a fertility clinic. Over twelve percent of pregnant persons reported moderate-to-severe anxiety symptoms. In univariate analysis, less than a college education (Pre-existing history of anxiety and socioeconomic factors likely exacerbated the impact of pandemic-related stressors on early pregnancy anxiety symptoms during the COVID-19 pandemic. Despite on-going limitations for in-person prenatal care administration, continued emotional health support should remain an important focus for providers, particularly when caring for less privileged pregnant persons or those with a pre-existing history of anxiety. The reported prevalence of anxiety during pregnancy varies widely (0.9% to 22.9%), depending on the population studied, with most studies reporting exclusively on symptoms in the second and third trimesters –3. CompaThe COVID-19 pandemic has resulted in widespread economic and social disruptions, all of which are likely to increase anxiety in susceptible individuals , 17. ThoA meta-analysis comprised of several retrospective studies found psychological stress to be a predictor of miscarriage . HoweverThis nationwide population-based study includes data from the University of California, San Francisco Assessing the Safety of Pregnancy in the Coronavirus Pandemic Prospective Cohort Study (UCSF ASPIRE). Only pregnant persons < 10 weeks’ gestation were eligible to enroll. Participants were recruited as two distinct samples: 1) a community sample and 2) Society for Assisted Reproductive Technology (SART) sample. The community sample included participants who acquired information about the study through either social media advertisement, the lay press, partnerships with online media companies focused on disseminating information to pregnant persons, traditional clinics providing prenatal care, or word of mouth. The SART sample comprised participants recruited from a network of fertility clinics nationwide with clinical encounters around the time of conception, allowing for precise gestational dating. Both cohorts of ASPIRE participants were prospectively followed throughout their pregnancy and postpartum courses. Prior to enrollment, written, informed consent was obtained. Participants completed baseline questionnaires regarding sociodemographic variables, history of COVID-19 exposure and/or infection, and mental health during the COVID-19 pandemic throughout their pregnancy. Study data were collected and managed using REDCap electronic data capture tools hosted at the University of California, San Francisco , 25. AddFrom May 2020 through February 2021, 5,207 pregnant persons across 48 states were enrolled into the UCSF ASPIRE study and completed the baseline questionnaire. Of respondents, 4,303 (82%) completed the Generalized Anxiety Disorder 7 (GAD-7) questionnaire and were included in this analytic sample. The mean age of participants was 33 years of age with over 65% of participants between age 31–40. The regional distribution was similar with the highest recruitment in the West (29.7%) and lowest from the Northeast (19.5%); otherwise, 24.8% and 25.9% were recruited from the Midwest and South, respectively. Close to 26% of participants were recruited from fertility clinics and comprised the SART cohort while roughly 74% were recruited through community.Upon enrollment, pregnant persons are asked to complete the GAD-7 questionnaire administered electronically. The GAD-7 is a validated 7-item scale which serves as a screening tool and severity indicator of generalized anxiety disorder and has been recommended to measure perinatal anxiety by the National Institute for Health and Care Excellence , 27. TheCharacteristics interrogated for their association with anxiety during pregnancy include: maternal age stratified based on the distribution of anxiety in our study , maternal race/ethnicity , maternal education , partner status , income , insurance status , working outside of the home , employment in health care , employment status , previous pregnancy and live birth , prior miscarriage , care through a fertility clinic this pregnancy , history of anxiety , and history of depression .We also investigated COVID-19 and economic-specific variables, including history of COVID-19 infection among oneself or household contacts , COVID-19 death among friends or family residing in the same household , job loss , reduced hours at work , inability to pay rent or mortgage , and worry about the health of self or loved ones during the COVID-19 pandemic . The Perceived Stress Scale (PSS) , a reliap = 0.10 level. Predictors in the first step included demographic characteristics, the second added obstetric and medical history variables, and the third added COVID-19 related characteristics. The third step in the model was implemented to determine how COVID-19 related characteristics contribute additionally to moderate-to-severe anxiety symptoms beyond known risk factors. All analyses were performed with Statistical Analysis Software (SAS®) version 9.4.This analytic sample was restricted to those who completed the GAD-7 baseline questionnaire. Descriptive characteristics were calculated for the variables of interest. Statistical analyses included univariate logistic regression with determination of predictors of moderate or severe anxiety symptoms (GAD-7: 10 or greater). A multivariate hierarchical logistic regression was performed to calculate adjusted odds ratios (aOR) and 95% confidence intervals (CI) predicting moderate-to-severe anxiety symptoms. Models were adjusted for variables selected as a covariate given its association with anxiety determined a priori (race/ethnicity) or significant in bivariate analyses to a We investigated the association between demographic factors and severity of anxiety symptoms, as estimated by GAD-7 scores. The percent of subjects with no to minimal, mild, moderate, and severe anxiety symptoms was 60.4%, 26.9%, 8.8%, and 3.8%, respectively Table . Rates oWe investigated the association between reproductive and medical history on anxiety symptoms. Persons with a history of a prior miscarriage were more likely to report greater anxiety symptoms. A history of anxiety and/or depression was associated with increased moderate-to-severe anxiety symptoms. Interestingly, persons who reported receipt of care through a fertility clinic this pregnancy had lower prevalence of moderate-to-severe anxiety symptoms Table .Finally, experiences and stressors unique to the COVID-19 pandemic were explored as a factor in increased moderate-to-severe anxiety. Pregnant persons reporting exposure to stressors such as job loss, reduced hours at work, or inability to pay rent or mortgage were more likely to report higher anxiety symptom severity. Furthermore, those who reported feeling very or extremely worried about the health of themselves or loved ones during the COVID-19 pandemic or the loss of a friend or family member residing in the same household due to infection with SARS-CoV-2 were more likely to report increased moderate-to-severe anxiety symptoms. Lastly, respondents who reported moderate-to-high perceived stress over the past month were more likely to report moderate-to-severe anxiety symptoms as compared to those with low perceived stress  and medical variables (history of anxiety or depression) were added Table . A pre-eCOVID-19 specific characteristics including having a household contact who died from COVID-19, job loss or reduced hours at work, inability to pay rent or mortgage, very or extreme worry about COVID-19, and perceived stress over the past month during the COVID-19 pandemic were entered into the third and final step of the model Table . This adWe sought to determine the prevalence of moderate to severe anxiety symptoms among nationwide pregnant persons at less than 10 weeks’ gestation during the COVID-19 pandemic. Between May 2020 through February 2021, over 4,300 pregnant persons from the ASPIRE prospective cohort responded to a validated measure of anxiety symptoms (GAD-7). We found that 12.6% of pregnant persons reported anxiety in the moderate-to-severe range, and the hierarchical regression showed that in addition to lower maternal education and a pre-existing history of anxiety, COVID-19 specific concerns or experiences were associated with these more severe symptoms.The lifetime prevalence of reported anxiety disorders is close to 30% with a greater risk exhibited among women as compared to men . Prior tLower education attainment and a pre-existing history of anxiety have previously been recognized as predictors of moderate-to-severe anxiety symptoms prior to the COVID-19 pandemic . SimilarRespondents who reported being very or extremely worried about personal or a close contact becoming infected with SARS-CoV-2 or losing a loved one during the COVID-19 pandemic were more likely to report more severe symptoms of anxiety even after accounting for preexisting sociodemographic and medical characteristics. These findings were similar to other reports showing the profound contribution of COVID-19 related concerns and experiences on developing anxiety related symptoms , 22, 31.The American Congress of Obstetricians and Gynecologists (ACOG) recently emphasized the importance of screening for mood disorders, including anxiety, given increasing awareness of the adverse clinical repercussions of untreated mood disorders in pregnancy . The panAn important strength of this work is the large cohort size and its national representation. Beyond the pandemic, this study represents one of the few studies to date to capture the prevalence and risk factors for anxiety symptoms during the first trimester. These results therefore represent novel information regarding a gestational time that has been historically difficult to access. We anticipate that future results from this longitudinal cohort will clarify the natural history and later pregnancy implications of anxiety initially identified in the first trimester. Nevertheless, there are several limitations to be considered as well. Although we utilized a gold-standard instrument for interrogating anxiety symptoms, questionnaire-based assessments lack the rigor of a clinical interview. Further, our cohort represents a population of persons interested and able to participate in a longitudinal seroprevalence study of COVID-19, and our recruitment scheme utilized both social media and fertility clinic-based recruitment and required enrollment prior to 10 weeks of gestation. As such, while this study is nationwide, it includes predominantly White, non-Hispanic women of higher socioeconomic status, thus, the generalizability of our findings should be considered with these factors in mind. We did not see a difference in anxiety symptoms based on the month enrolled into the cohort. However, as burden from the COVID-19 has differed by region throughout this period, it is possible there is an association between regional surge and anxiety symptoms we were unable to disentangle. While this could be a potential factor associated with the presence of anxiety symptoms, we do not feel this confounded the associations otherwise seen in this analysis. We sought to ascertain if working on-site differentially impacted anxiety symptoms when compared to employment that allowed some or completely virtual options. While this variable was not a significant predictor in our analysis, we acknowledge the structure of the question may have impacted any potential association. Finally, our findings likely do not accurately reflect the experience of persons with less interest in or capacity for research participation or the experience of those who did not become aware of their pregnancy until after 10 weeks.A prior history of anxiety, low maternal educational attainment and concerns specific to the COVID-19 pandemic were significant predictors of moderate-to-severe anxiety in the ASPIRE cohort. Continued emotional health support should remain an important focus for providers, particularly when caring for those who are underserved, have a preexisting history of anxiety, and early in pregnancy when care may be limited. With limitations in in-person prenatal care as a function of the COVID-19 pandemic, continued attention to mental health screening and appropriate intervention should remain an important focus for providers. Furthermore, future studies are essential to explore the continued impact of the pandemic on antenatal and postnatal anxiety symptoms especially among the most vulnerable populations.


At discharge, NT-proBNP levels were lower and cumulative urine output greater in the empagliflozin group versus the basal-bolus insulin group . Patients who continued empagliflozin had a lower total number of hypoglycaemic episodes . No differences were observed in adverse events, length of hospital stay, or in-hospital deaths. For patients with acute heart failure, an in-hospital antihyperglycaemic regimen that includes continuation of empagliflozin achieved effective glycaemic control, lower NT-proBNP, and greater urine output. It was also safer, as it reduced hypoglycaemic episodes without increasing other safety endpoints.There is little evidence on the use of sodium−glucose cotransporter 2 inhibitors in hospitalised patients. This work aims to analyse the glycaemic and clinical efficacy and safety of empagliflozin continuation in patients with type 2 diabetes hospitalised for acute decompensated heart failure. This real-world observational study includes patients treated using our in-hospital antihyperglycaemic regimens  between 2017 and 2020. A propensity matching analysis was used to match a patient on one regimen with a patient on the other regimen. Our primary endpoints were the differences in glycaemic control, as measured via mean daily blood glucose levels, and differences in the visual analogue scale dyspnoea score, NT-proBNP levels, diuretic response, and cumulative urine output. Safety endpoints were also analysed. After a propensity matching analysis, 91 patients were included in each group. There were no differences in mean blood glucose levels (152.1 ± 17.8 vs. 155.2 ± 19.7 mg/dL,  Heart failure (HF) is one of the leading causes of hospital admissions around the world and entails a high risk of early postdischarge mortality and rehospitalisation ,2.In recent years, multiple randomised clinical trials have reported that sodium−glucose cotransporter 2 (SGLT2) inhibitors significantly reduce hospitalisations for acute decompensated HF in patients with type 2 diabetes (T2D) ,4,5,6,7.In non-critically ill patients with T2D, a multidose insulin regimen that involves once-daily basal insulin and bolus of rapid-acting insulin before meals ,11 and, We conducted a real-world observational study on patients with T2D admitted for acute decompensated HF to 4 hospitals in Málaga, Spain  from January 2017 to December 2020. Investigators from each hospital reviewed each patient’s electronic medical records in order to collect patient data.Acute HF was defined as all of the following: dyspnoea at rest or on minimal exertion, signs of congestion , N-terminal pro-brain natriuretic peptide (NT-proBNP) ≥1400 pg/mL , and intravenous treatment with loop diuretics.2, clinically-relevant liver disease or cirrhosis, blood dyscrasias or any disorders causing haemolysis or unstable red blood cells, gastrointestinal obstruction, those expected to be without oral intake, use of artificial nutrition , urinary tract infection, genital infection, perineal necrotizing fasciitis, acute peripheral vascular disease, and pregnant or nursing (lactating) women. The choice of which regimen to use is made by physicians according to their clinical judgment.Non-critically ill patients with T2D hospitalised for acute decompensated HF and treated with empagliflozin for at least 3 months prior to the hospitalisation were selected. According to our in-hospital antihyperglycaemic protocol, patients can be treated with two possible regimens: a basal-bolus insulin regimen or an empagliflozin-basal insulin regimen. The basal-bolus insulin regimen is the conventional glucose-lowering treatment used in the hospital setting and is recommended for all patients as a standard of care regardless of their admission blood glucose level. This regimen includes the discontinuation of empagliflozin and the initiation of once-daily basal insulin , administered at 04:00 p.m.) and rapid-acting insulin analogues before meals  or insulin aspart . Alternatively, patients with an admission blood glucose (BG) level of less than 250 mg/dL who do not meet any exclusion criteria have the option to continue with empagliflozin . This regimen involves a single dose at the same dose as prior to hospitalisation (10 or 25 mg) administered at 09:00 a.m. in addition to a once-daily basal insulin , administered at 04:00 p.m.). The exclusion criteria for empagliflozin use are: signs of ketoacidosis and/or hyperosmolar hyperglycaemic state, type 1 diabetes, concomitant hospital treatment with a systemic glucocorticoid, expected admission to an intensive care unit, planned cardiac surgery, acute renal function impairment with an estimated glomerular filtration rate (based on the Chronic Kidney Disease Epidemiology Collaboration formula )  ≥ 45 mL/The insulin dose is calculated according to admission BG levels, age, and serum creatinine and is modified during hospitalisation when required . When supplemental rapid-acting insulin is required before meals and bedtime, the dose is calculated according to BG levels, total daily insulin units, and the patient’s weight. Fasting, preprandial, and bedtime capillary BG levels are measured using a point-of-care glucose meter. Additionally, BG levels are measured any time a patient experiences symptoms of hypoglycaemia or when requested by the healthcare provider. Level 1 hypoglycaemia is defined as a measurable BG level <70 mg/dL and ≥54 mg/dL, level 2 hypoglycaemia as a measurable BG level <54 mg/dL, and level 3 hypoglycaemia as a severe event characterised by an altered mental and/or physical status requiring assistance, according to the American Diabetes Association criteria . When paOur primary endpoint was to analyse the differences in glycaemic control, as determined by mean daily BG levels; BG levels at mealtime and bedtime; BG levels 100–140 mg/dL, 140–180 mg/dL, and 180–250 mg/dL; number and day of treatment failures; total daily insulin dose ; and number of daily insulin injections between the empagliflozin-basal insulin and basal-bolus insulin regimens in patients with T2D admitted for acute decompensated HF. In addition, we analysed the differences between regimens in regard to the visual analogue scale (VAS), dyspnoea score and the NT-proBNP levels from baseline (at admission) to discharge; diuretic response, defined as weight loss (kilograms) per 40 mg furosemide or equivalent at discharge; and cumulative urine output during hospitalisation.Safety endpoints included total adverse events , worsening HF , adverse events that lead to the discontinuation of empagliflozin (excluding the discontinuation of empagliflozin due to treatment failure), hypoglycaemic episodes , length of hospital stay and in-hospital deaths.Statistical analyses were performed using SAS for Windows, version 9.3 , and SPSS Statistics for Windows, version 15.0 .In order to match each patient who started on the basal-bolus insulin regimen with a patient who started on the empagliflozin-basal insulin regimen in a 1:1 manner, a propensity score with a caliper of 0.2 and a greedy matching algorithm were used. The probability of starting the empagliflozin-basal insulin regimen  was estimated using a logistic regression model that included variables that could have affected treatment assignment or outcomes as independent variables  functional classification; left ventricular ejection fraction; principal cause of heart failure; prior hospitalization for heart failure, heart failure medication; amount of time the patient has had T2D; BG, serum creatinine, estimated glomerular filtration rate, NT-proBNP, and transaminase levels at admission; body mass index; and at-home treatment). In order to assess the adequacy of propensity matching, we used the standardised difference (SD) of patient characteristics after matching. A significant imbalance in the group was considered to be present if an SD between baseline variables of higher than 10% was found.p < 0.05. Multiple comparisons across different days of therapy were adjusted conservatively using Tukey’s adjustment.Baseline characteristics were analysed using descriptive statistics. Continuous and categorical variables were shown as means ± standard deviation and as absolute value and percentage, respectively. The hypoglycaemia incidence rate per 100 patient-years was calculated. The differences between groups were determined using the two-sample Student’s t-test or the Mann-Whitney U test for continuous variables and Pearson’s χ2 test for categorical variables. Values were considered to be statistically significant when Only patients who had previously given consent for their medical records to be used for medical research were included. Data confidentiality and patient anonymity were maintained at all times, in accordance with Spanish regulations on observational studies. Patient identifying information was deleted before the database was analysed; it is not possible to identify patients on an individual level either in this article or in the database. This study was carried out in accordance with the Declaration of Helsinki and was approved by the Institutional Research Ethics Committee of Málaga on 27 October 2016 (Ethics Committee code: REDIME 27-10-2016).A total of 347 patients with T2D hospitalised for acute decompensated HF and treated with empagliflozin before hospitalisation were included. Of them, 196 (56.5%) discontinued empagliflozin and started the basal-bolus insulin regimen and 151 (43.5%) continued empagliflozin at the same dose as prior to hospitalisation  in combination with basal insulin. After propensity matching, 91 patients were included in each group. In the empagliflozin group, 40 patients had a 10-mg dose and 51 patients had a 25-mg dose before hospitalisation. A flow chart for patient inclusion for both regimens is shown in The baseline clinical characteristics of patients were well balanced between groups following the propensity matching analysis, with standardised differences of <10%. Before the propensity matching analysis, patients who continued the empagliflozin were younger and had a higher NYHA functional class. These data are shown in In regard to the glycaemic control, there were no differences between the basal-bolus insulin and the empagliflozin-basal insulin regimen groups in mean daily BG levels during hospitalisation; BG levels at mealtime and bedtime; BG levels 100–140 mg/dL, 140–180 mg/dL, and 180–250 mg/dL; and the number and day of treatment failures after the propensity matching analysis. However, the total insulin dose and number of injections per day during hospitalisation were significantly lower in the empagliflozin-basal insulin group compared to the basal-bolus insulin group. Total basal and supplemental rapid-acting insulin doses did not differ significantly between the treatment groups. Before matching, patients managed with the empagliflozin-basal insulin regimen had a higher mean BG level during hospitalisation, mean BG level before lunch, a mean BG 180–250 mg/dL, and number of treatment failures than those managed with the basal-bolus insulin regimen. Similar to the data found on the post-propensity matching analysis, patients managed with the basal-bolus insulin regimen received more total insulin and a greater number of injections per day. No differences were observed in total basal and supplemental rapid-acting insulin doses. These data are summarised in p = 0.148), but NT-proBNP levels were lower in the empagliflozin-basal insulin group than in the basal-bolus insulin group . Moreover, although no significant difference was found in the diuretic response between groups , the cumulative urine output was significantly greater in patients treated with empagliflozin compared with basal-bolus insulin during the hospitalisation . These results are shown in p = 0.348). No significant differences were observed in blood pressure levels between groups.The VAS dyspnoea score and NT-proBNP levels declined during hospitalisation. At discharge, there was no difference in the VAS dyspnoea score between groups , length of hospital stay , or in-hospital deaths between groups after the propensity matching analysis. Six patients (6.6%) had adverse events that led to the discontinuation of empagliflozin. The empagliflozin-basal insulin group had a lower total number of hypoglycaemic episodes, patients with 1 or ≥2 episodes, hypoglycaemia incidence rate, and patients with any level 1 hypoglycaemia. Before matching, patients on the basal-bolus insulin regimen had more cardiovascular events and instances of worsening HF, though the total number of adverse events did not differ between the groups. All hypoglycaemic episodes analysed were significantly more frequent in patients on the basal-bolus insulin regimen than those on the empagliflozin-basal insulin regimen. The length of hospital stay and number of in-hospital deaths were similar between the treatment groups. Safety endpoints are summarised in This real-world study found that in patients with T2D hospitalised for acute decompensated HF, the in-hospital continuation of empagliflozin in combination with basal insulin was as efficacious in regards to glycaemic control as a conventional multidose basal-bolus insulin regimen. In addition, the empagliflozin regimen increased the cumulative urine output and reduced the NT-proBNP levels during hospitalisation but led to no differences in the VAS dyspnoea score or diuretic response between groups. Finally, the empagliflozin-basal insulin regimen was found to be safer than the conventional basal-bolus insulin regimen, as there were fewer hypoglycaemic episodes. No differences were observed in adverse events, length of hospital stay, or in-hospital deaths.To our knowledge, this work is the first real-world study addressing the safety and glycaemic and clinical efficacy of empagliflozin continuation during the hospitalisation of patients with acute decompensated HF. To date, two randomised controlled clinical trials with small sample sizes have evaluated the effects of empagliflozin on clinical outcomes in patients hospitalised for acute decompensated HF ,21. The Unlike chronic HF, there are currently no therapies available that improve the prognosis of acute decompensated HF. The use of loop diuretics to reduce congestion remains the mainstay of acute HF treatment . SeveralBoth randomised clinical trials on this subject were focused on clinical endpoints and no data on glycaemic control were reported ,21, despRegarding safety endpoints, both the Damman et al.  and TamaSGLT2 inhibitors have shown clear benefits in HF hospitalisations in patients with ,4,5,6,7 Although our findings are important, this study has several limitations. First, given the retrospective nature of our data, the use of a propensity matching analysis cannot rule out the possibility of unmeasured confounding factors. Second, continuation of empagliflozin in patients who are hospitalised is not fully implemented in our area. In fact, most patients were managed with the conventional basal-bolus insulin regimen and discontinued empagliflozin. Third, there was no standardised protocol for the treatment of acute decompensated HF and no protocol for diuretic therapy, which could have influenced our findings. Fourth, due to the relatively low number of events and complications, their relationship to the antihyperglycaemic regimen could not be conclusively determined. Fifth, only empagliflozin continuation was evaluated in this study. The reasons behind this choice are that a significant percentage of patients are treated with empagliflozin in our area and we wanted to evaluate its efficacy and safety in isolation, without the interference of other SGLT2 inhibitors. An interesting future line of research would be an evaluation of continuing other SGLT2 inhibitors during hospitalisation. Lastly, we only included patients admitted for acute decompensated HF, but the efficacy and safety of empagliflozin could be assessed in other groups of patients hospitalised for other cardiovascular diseases. Further research is required in order to confirm these results and provide more evidence. We found that an in-hospital antihyperglycaemic regimen that includes the continuation of empagliflozin achieved effective glycaemic control and was associated with greater cumulative urine output and lower NT-proBNP levels than a conventional basal-bolus insulin regimen in patients with T2D hospitalised for acute decompensated HF. In addition, the empagliflozin regimen was found to be safer, as there were fewer hypoglycaemic episodes and no differences in adverse events, length of hospital stay, or in-hospital deaths. Large, randomised trials with SGLT2 inhibitors are required to provide more evidence on the efficacy and safety of SGLT2 inhibitors in patients with acute decompensated HF.


NTA in fluorescence mode (F-NTA) demonstrated that only 9.5% of all vesicles detected in scatter mode exposed phosphatidylserine, resulting in good agreement of LA+ EVs for CytoFLEX LX and F-NTA. Since certain functional characteristics, such as the exposure of pro-coagulant phosphatidylserine, are not equally displayed across the entire EV size range, our study highlights the necessity of indicating the size range of EVs detected with a given approach along with the EV concentration to support the comparability between different studies.Growing interest in extracellular vesicles (EVs) has prompted the advancements of protocols for improved EV characterization. As a high-throughput, multi-parameter, and single particle technique, flow cytometry is widely used for EV characterization. The comparison of data on EV concentration, however, is hindered by the lack of standardization between different protocols and instruments. Here, we quantified EV counts of platelet-derived EVs, using two flow cytometers  and nanoparticle tracking analysis (NTA). Phosphatidylserine-exposing EVs were identified by labelling with lactadherin (LA). Calibration with silica-based fluorescent beads showed detection limits of 300 nm and 150 nm for Gallios and CytoFLEX LX, respectively. Accordingly, CytoFLEX LX yielded 40-fold higher EV counts and 13-fold higher counts of LA EVs are subcellular fragments that originate from the endosomal compartment or are shed from the plasma membrane of virtually all cell types of the human body under both, physiological and pathological conditions ,2,3. TheThe generic term “extracellular vesicles” encompasses individual vesicle subpopulations, which display overlapping features, despite their heterogeneity ,7. SmallIsolation and characterization of individual EV populations require a combination of different methods  and stilFlow cytometry is well-established for the characterization of EVs directly in body fluids because of its high throughput and its ability to label and discriminate EVs of different cellular origin. Still, current flow cytometers are not capable of fully resolving individual EVs on the basis of light scatter , as cryoDespite these limitations, EV detection using flow cytometry is according to the current estimates used in 90% of all studies in the field of EV research ,13, partNTA is suited for the rapid assessment of size and concentration of nanoparticles ranging from 40–1000 nm in diameter ,15. It cHere, we employed flow cytometry and differential S-NTA/F-NTA for the characterization of different batches of platelet-derived EVs. Using two different flow cytometers , we compared total EV counts as well as the amount of phosphatidylserine-exposing EVs and related these data to results obtained with differential S-NTA/F-NTA, revealing vast differences in EV counts for the different techniques and instruments.+CD41+ EVs/µL as compared to the Gallios flow cytometer  are likely due to donor-dependent variations in the concentration of blood lipids, e.g., levels of cholesterol and/or triglycerides. The FT-IR spectra for platelet-derived EVs were similar to previously published patterns for monocytic EVs [−1), amide I (1652 cm−1), and amide II (1546 cm−1). Peaks at 2923 and 2852 cm−1 corresponded to asymmetric and symmetric methylene (C–H) stretching. FT-IR spectroscopic protein-to-lipid ratios were calculated from the data according to Mihaly et al. [We employed FT-IR spectroscopy as additional label-free measurement mode to characterize EVs. ytic EVs  or red bytic EVs . The stry et al.  and yielIn our current study, we compared the ability of two conventional flow cytometers and of nanoparticle tracking analysis to quantify platelet-derived EVs. NTA detects EVs down to a size of about 50 nm, whereas the flow cytometers used in our study, Gallios and CytoFLEX LX, have a detection limit of about 300 and 150 nm, respectively ,21. The + EVs between the two instruments. While more than 90% of all events detected in the EV gate with the Gallios instrument were LA+ and thus exposed phosphatidylserine on their surface, CyoFLEX LX identified only 64% of LA+ events in the EV gate. This is most likely explained by the fact that the detection of EVs using Gallios is limited to larger EV populations, which are preferentially derived from the plasma membrane and, in contrast to smaller EVs derived from the endosomal compartment, expose phosphatidylserine on their outer membrane leaflet. Our findings underline that reported EV concentrations should always be accompanied by information on the minimal detectable EV size for a given instrument. This is crucial to estimate the percentage of certain EV subpopulations, and is particularly essential wherever a functional trait  is not uniformly distributed in a population, but is preferentially associated with vesicles of a particular size range .In flow cytometry, we evaluated both, total EV counts (events/µL) and counts of EVs exposing phosphatidylserine, which were identified by staining with lactadherin-FITC. In agreement with its enhanced ability to detect smaller EVs, CytoFLEX LX yielded 40-fold higher total EV counts as compared to the Gallios instrument. Our study also revealed differences in the percentage of LA+ EV counts for CytoFLEX LX (lactadherin-FITC) and NTA (lactadherin-AF555).Comparing flow cytometry and NTA, the total events detected with NTA in scatter mode exceeded flow cytometric EV counts by more than one (CytoFLEX LX) or two  orders of magnitude, respectively. This is in good agreement with previous data reported by van der Pol et al. who found 15 times lower EV counts with flow cytometry as compared to NTA , which wAccording to F-NTA analysis following labelling of EVs with CMO vs. lactadherin-AF555, 36% of all particles detected in scatter mode were stained with CMO, a fluorescent plasma membrane label composed of amphipathic molecules comprising a lipophilic moiety for membrane loading and a negatively charged hydrophilic dye for anchoring of the probe in the plasma membrane . This in+ particles in comparison to CMO+ particles supports the findings from flow cytometry that phosphatidylserine is predominantly present on larger EV populations. Still, EV sizes obtained with F-NTA might also be affected by the staining procedure itself, since antibody or dye bound to the EV surface can affect the Brownian motion of the particles [The smaller size of particles obtained by S-NTA as compared to F-NTA indicates the detection of smaller non-EV contaminants, such as protein aggregates or lipoproteins, in scatter mode. We are currently assessing whether further purification of EV samples by size exclusion chromatography results in the depletion of co-isolated protein complexes/lipoproteins, to reveal a more accurate size distribution of the “true” EV population in scatter mode. The increased size of LAarticles .While applying the same sample dilution for both, scatter mode and fluorescent mode would be optimal to avoid inaccuracies caused by pipetting, this approach is only applicable for EV samples derived from matrices with comparatively low complexity, such as EVs isolated from cell culture supernatants. In the case of complex sample matrices, such as platelet concentrate, however, measurements in scatter mode require a much higher dilution than in fluorescent mode due to the background caused by plasma protein aggregates or lipoproteins. To limit inaccuracies related to pipetting in our study, we performed all measurements at least in triplicates .−1) and C-H (2700–3000 cm−1) stretching vibrations, the spectroscopic protein-to-lipid ratio was proposed as sample quality parameter [As a supplement to our study, we evaluated the suitability of FT-IR spectroscopy to determine the protein-to-lipid ratio of platelet-derived EVs. Whereas FT-IR is not commonly applied for EV quantification, it has been used to characterize the composition of EVs of different cellular origin, e.g., prostate cancer cells , monocytarameter . We obtaarameter  or from arameter . HoweverIt would be of interest to extend this study to other flow cytometers, particularly to instruments from other manufacturers, and to compare different NTA devices from ZetaView (PMX-110 vs. PMX-120 with higher sensitivity) as well as from other figureiers . Still, taking into account that pre-analytical parameters including storage of EV samples, particularly of platelet-derived EVs, induce additional EV release from residual platelets, we limited our investigations to the devices available in our laboratory, to avoid bias caused by sample storage and transportation.The accurate characterization of EVs using well-defined protocols is crucial for subsequent functional studies, but the phenotypical characterization of the whole EV spectrum is difficult to attain. This is especially challenging for the characterization of EVs from highly complex matrices such as whole blood, which contain lipoproteins and protein aggregates with overlapping characteristics  ,16,22. WIn conclusion, our study revealed profound differences in EV concentrations obtained with two different conventional flow cytometers and with NTA. Different vesicle concentrations were primarily caused by differences between the minimal detectable EV sizes in flow cytometry, and, additionally, by the detection of non-vesicular light scattering structures in NTA. Since certain functional characteristics, such as the exposure of pro-coagulant phosphatidylserine, are not equally displayed across the entire EV size range, our study highlights the necessity of indicating the size range of EVs detected with a given approach along with the EV concentration to support the comparability between different studies.+; Macopharma, Tourcoing, France) at a ratio of 80% SSP+ and 20% plasma, and were used for EV isolation within 2 days.Medical grade platelet concentrates from healthy donors were provided by the Clinic for Blood Group Serology and Transfusion Medicine, Medical University of Vienna, Vienna, Austria, as approved by the local ethics committee (ECS2177/2015). Prior to sample acquisition, written informed consent was obtained from all donors. Samples were collected in a blood bank setting using a Trima Accel R automated blood collection system . Platelet concentrates were stored in polyolefin bags in storage solution for platelets  to deplete platelets and debris. EVs were pelleted at 20,000× g  using a Sorvall Evolution RC ultracentrifuge equipped with an SS-34 rotor . The pellet was washed with sterile phosphate buffered saline (PBS) without calcium and magnesium , re-centrifuged at 20,000× g , and re-suspended in 200 μL PBS. The protein content was quantified using the DC Protein Assay . Samples were normalized to a protein concentration of 4 mg/mL, aliquoted, and stored at −80 °C until further use. Eight batches from different donors were used for all measurements.EVs were enriched from platelet concentrates by differential centrifugation, as previously described . Plateleg for 10 min at RT prior to use to remove aggregates. Stained samples were diluted 5-fold in PBS and analyzed on a Gallios and a CytoFLEX LX flow cytometer . All fluorochrome conjugates used for flow cytometry are specified in EV suspensions were diluted in filtered PBS  to a protein concentration of 1 µg/mL. Aliquots of 100 µL of the diluted samples were stained for 15 min at RT in the dark with 83 ng fluorescein isothiocyanate-conjugated lactadherin  as marker of phosphatidylserine, as well as with 100 ng phycoerythrin cyanin 7 (PE-PC7)-conjugated anti-CD41 antibody  as platelet marker. To avoid swarm detection, the optimal sample protein concentration was determined as specified in EV counts and size distribution were assessed by NTA . For measurements in scatter mode, isolated platelet-derived EVs were diluted 5000-fold  in 0.1 μm filtered PBS. Measurements were performed in triplicates at RT at a camera sensitivity of 80%, counting an average of 1000 tracks with 15 frames per second. In addition to scatter mode measurements, fluorescence-based analysis was performed after staining with the membrane dye CMO  or with LA-AF555 . All dyes and fluorochrome conjugates used for F-NTA are specified in 3 detector and a MIRacle™ single reflection Zinc Selenide ATR (ZnSe) accessory . For spectral manipulations, the Spectrum 10  and OMNIC 8.1.0.10  software versions were used. The detailed protocol for FT-IR measurements is described in the Text S1.All measurements were performed on a Spectrum Two FT-IR Spectrometer  equipped with a LiTaOt-test.Statistical analysis was performed using GraphPad Prism, version 8.2 . Data are presented as mean ± standard deviation (SD). One-way repeated measures ANOVA or two-way repeated measures ANOVA followed by Sidak’s multiple comparisons test were used to compare three groups. Statistical significances between two groups were determined by the paired


Asfotase alfa is the only approved treatment that can normalize mineralization in patients with hypophosphatasia (HPP). Its interference in alkaline phosphatase (ALP) dependent immunoassays has been reported. Patients and Methods. Three patients with HPP treated with asfotase alfa were included. Thyroid hormone levels measured using five different immunoassays with or without ALP as a labeling enzyme during asfotase alfa treatment were evaluated.  To describe thyroid function tests interfered with by asfotase alfa and elucidate the underlying mechanism.  After the initiation of asfotase alfa, three HPP patients showed low free triiodothyronine (FT3) and free thyroxine (FT4) measured with AIA-2000 , an enzyme immunoassay system that uses ALP as a labeling enzyme, but their thyroid-stimulating hormone (TSH) levels were within the normal range. The other CLEIA system using ALP as a label, AIA-CL2400 , and ALP-independent immunoassay systems demonstrated normal FT3 and FT4 levels. These data suggested that although the thyroid function of these three patients was normal, asfotase alfa interfered with the thyroid hormone measurements made with AIA-2000. AIA-2000 and AIA-CL2400 adopted one-step and delayed one-step measurements, respectively, and the same antibody was used for both immunoassays. However, asfotase alfa may be absorbed on the magnetic beads used in the AIA reagent with the AIA-2000 system but not absorbed on the microparticles used in AIA-CL2400.  Clinicians should be aware of the possible interference in thyroid function measurements by adopting specific types of immunoassays in asfotase alfa-treated HPP patients. Mutati) . Because disease , 7. HPP  disease . Among J disease . The homAsfotase alfa  consists of alkaline phosphatase (ALP) and Fc regions of the antibody conjugated with deca-aspartate, and it is the only treatment method available that can improve impaired mineralization in the bone among patients with HPP and has become available in many countries, including Japan, Canada, the United States, and the European Union .ALP is also commonly used as a label substance in enzyme-linked immunosorbent assays (ELISAs) for routine measurements of hormones and other biomarkers. In immunoassays that use ALP for signal amplification, ALP hydrolyzes substrates (paranitrophenylphosphate (pNPP), 4-methylumbelliferyl phosphate phosphatase (4-MUP), nitro blue tetrazolium salt and 5-bromo-4-chloro-3-indolyl-phosphate (NBT/BCIP), or nicotinamide adenine dinucleotide phosphate (NADPH)) and creates a color/fluorescence change that is measured with a spectrophotometer. In the sandwich ELISA system, the amount of enzyme is directly proportional to the amount of measured biomarkers. On the other hand, in the competitive ELISA, the quantity of antigen in the sample is inversely correlated with the amount of enzymatic activity.β-human chorionic gonadotropin, and oxytocin [Because asfotase alfa contains an ectodomain region of ALP that is similar to the ALP used in some ELISAs, asfotase alfa has been reported to have the potential to interfere with ELISAs using ALP-labeled tracers. To date, a few case reports have shown that asfotase alfa may interfere with several laboratory tests, such as testosterone, cardiac troponin I, oxytocin –14. In tThree patients with HPP who regularly visited our institute were included in the present study. All procedures were performed in accordance with the ethical standards of the Declaration of Helsinki and approved by the institutional ethical board of the University of Tokyo Hospital (refs. 2879 and 11221), and all participants provided written informed consent.Patient data including age, diagnosis, the result of the genetic analysis, timing of the initiation of asfotase alfa, and thyroid functions were extracted retrospectively.Thyroid-stimulating hormone (TSH), free triiodothyronine (FT3), and free thyroxine (FT4) in the three adult participants with HPP with or without asfotase alfa were measured in five immunoassay systems: AIA-2000 , AIA-CL2400 , AccuraSeed , ARCHITECT i2000SR , and Cobas 8000 e801  . AIA-200The first case was a 67-year-old woman who presented with periarticular calcification and was diagnosed with adult-onset HPP with iliac crest biopsy that demonstrated impaired mineralization. Mutational analysis showed a heterozygous pathogenic single nucleotide deletion in ALPL, c.1559delT (p.Leu520ArgfsX86). Replacement therapy with asfotase alfa (240 mg/week: 4 mg/kg/week) has been administered since the patient was 65 years old.Case 2 was a 23-year-old man with infantile severe HPP whose genetic test revealed a compound heterozygous mutation in ALPL, c.670A > G (p.Lys224Glu) and c.1276G > T (p.Gly426Cys). He has been treated with asfotase alfa (240 mg/week: 6 mg/kg/week) since 17 years of age.The patient was a 22-year-old woman with benign prenatal HPP. Genetic testing revealed that she had compound heterozygous mutations in ALPL, c. 979T > C (p. Phe310Leu) and c.1559delT (p.Leu520Argfs86). Treatment with asfotase alfa (240 mg/week: 6 mg/kg/week) was initiated at the age of 19 years.. All immunoassay systems other than AIA-2000 demonstrated normal TSH, FT3, and FT4 levels, which indicated normal thyroid function in the three participants with HPP undergoing asfotase alfa treatment and interference between asfotase alfa and AIA-2000 in an antibody-independent manner  was evaluated at the laboratory at the University of Tokyo Hospital. Of all three cases, normal thyroid functions were confirmed with an ALP-mediated measuring/signaling system (AIA-2000) before the initiation of asfotase alfa in cases 1 and 3, and there was no record of thyroid function for case 2 before treatment. However, thyroid function measurements using the AIA-2000 system after the initiation of asfotase alfa  revealed abnormally low FT3 and FT4 levels with concomitant inappropriately normal TSH levels without any typical symptoms of hypothyroidism . The imm. All immt manner .In the present report, a thyroid function test using AIA-2000, an immunoassay system that uses ALP as a labeling enzyme, revealed abnormally low levels of FT3 and FT4 despite normal TSH levels in three patients with HPP treated with asfotase alfa. On the other hand, a thyroid function test using AIA-CL2400, another immunoassay system using the same antibodies and ALP as a label, and an ALP-independent immunoassay analyzer revealed normal FT3 and FT4 levels. Asfotase alfa may be absorbed on the magnetic beads used in the AIA-2000 system, which explains the interference observed exclusively in thyroid function tests performed with AIA-2000 but not AIA-CL2400.Occasionally, different substances interfered with the results of some immunoassay systems by various mechanisms, such as analyte-independent interference or analyte-dependent interference , 15. Anaβ-human chorionic gonadotropin when measured with an assay system that used ALP as a substrate [Two mechanisms that explain the residual asfotase alfa after washing have been reported. The first mechanism was the inadequate washing of unbound analyte. Laboratory tests of thyroid hormones are well known to be affected by exogenous autoantibodies or medications . Additioubstrate . The secubstrate .in vitro assays that the magnetic beads from AIA-2000 exhibited a high affinity for asfotase alfa, whereas the magnetic microparticles used in AIA-CL2400 did not have any affinity for asfotase alfa. No interference between ALP and magnetic beads or between ALP and magnetic microparticles was observed, suggesting specific interference between asfotase alfa and magnetic beads used in AIA-2000 (data not shown). However, the detailed study protocol and actual measurement values cannot be disclosed under a confidentiality agreement.Although the two abovementioned mechanisms have been reported, both mechanisms failed to explain the discrepancy in thyroid hormone levels between the two analyzers that utilized ALP for signal enhancement. AIA-2000 and AIA-CL2400 adopted one-step and delayed one-step measurements, respectively, and both measurement methods had similar washing protocols. Additionally, both analyzers used the same antibody set. Given that FT3 and FT4 measurements obtained with the AIA-2000 and AIA-CL2400 systems utilized magnetic beads with primary antibody and magnetic microparticles with primary antibody, respectively, this difference could explain the discrepancy in FT3/FT4 measurements . In factAlthough this study revealed the direct absorption of asfotase alfa to magnetic beads as a novel potential mechanism explaining the altered thyroid hormone levels observed in specific types of ALP-based immunoassay systems, there were some limitations. First, detailed measurement methods, including precise structures of magnetic beads in AIA-2000 and magnetic microparticles in AIA-CL2400, were not fully disclosed due to the manufacturer's regulations, and the details of the experimental results comparing the affinities between asfotase alfa and magnetic beads and magnetic microparticles performed by the manufacturer could not be disclosed in this study. Second, only two of the ALP-based analyzers were included in the present study, and the same interfering mechanism described in the present study was not validated in any of the other ALP-based analyzers. Further studies are therefore warranted to analyze asfotase alfa interference in thyroid function tests using other ALP-based assay systems.The present study revealed that asfotase alfa could affect some of the ALP-based immunoassays using specific types of magnetic beads that have an affinity for asfotase alfa. Particularly, this was the first report describing the novel interference between asfotase alfa and the thyroid function test. Clinicians should be aware of the possible interference in thyroid function measurements by adopting specific types of magnetic beads in asfotase alfa-treated HPP patients.


Unverified information concerning COVID-19 can affect mental health. Understanding perceived trust in information sources and associated mental health outcomes during the COVID-19 pandemic is vital to ensure ongoing media coverage of the crisis does not exacerbate mental health impacts. A number of studies have been conducted in other parts of the world to determine associations between information exposure relating to COVID-19 and mental health. However, the mechanism by which trust in information sources may affect mental health is not fully explained in the developing country context. To address this issue, the present study examined associations between perceived trust in three sources of information concerning COVID-19 and anxiety/stress with the mediating effects of COVID-19 stress in Bangladesh. An online cross-sectional study was conducted with 744 Bangladeshi adults between 17 April and 1 May 2020. Perceived trust in traditional, social, and health media for COVID-19 information, demographics, frontline service status, COVID-19-related stressors, anxiety (GAD-7), and stress (PSS-4) were assessed via self-report. Linear regression tested for associations between perceived trust and mental health. Mediation analyses investigated whether COVID-19-related stressors affected perceived trust and mental health associations. In fully adjusted models, more trust in social media was associated with more anxiety  and stress , while more trust in traditional media was associated with more anxiety  but less stress . Mediation analyses showed that COVID-19-related stressors partially explained associations between perceived trust and anxiety. These findings suggest that trusting social media to provide accurate COVID-19 information may exacerbate poor mental health. These findings also indicate that trusting traditional media  may have stress-buffering effects. We recommend that responsible authorities call attention to concerns about the trustworthiness of social media as well as broadcast positive and authentic news in traditional media outcomes based on these results. The COVID-19 disease has posed a substantial humanitarian problem raising severe public health concerns in all nations. On 31 December 2019, the first cases of COVID-19 were officially reported, though some Wuhan authorities indicated that the first cases occurred even earlier, between 12 and 29 December. COVID-19 was then declared a global pandemic on 11 March 2020 . Of incrEarlier studies have reported that spreading misinformation can cause poor mental health outcomes, such as fear, anxiety, stigma, and threatening behavior ,9,10,11.The lack of reliable information about COVID-19 is of particular concern given its role as a prerequisite for mitigating the health impacts of the virus ,20. RegaMisinformation on unregulated media channels was especially noticeable in the early stages of the COVID-19 pandemic. The popular social networking site Facebook documented around 90 million pieces of content between March and April 2020 that spread fake news regarding COVID-19 . A studyMisinformation during the COVID-19 pandemic has been connected to myriad mental health concerns . Panic rSuch health impacts of media consumption during the COVID-19 pandemic and other crises might be explained in part by the Differential Susceptibility to Media Effects Model (DSMM) . This moThe first case of COVID-19 in Bangladesh was confirmed on 8 March 2020. On 18 March 2020, Bangladesh reported its first death from COVID-19 . To prevCentella asiatica) would prevent COVID-19 infection. Correspondingly, the market price for this “longevity herb” rose fives time higher than its regular price [Rubus sp.) consumption. Half believed that regularly eating garlic could be a remedy for COVID-19 infection. Such misleading information created some confusion and panic among people as well as challenges for the Bangladesh Government [Bangladesh has 0.1 billion internet users, but many of these users lack basic digital literacy . Furtherar price . A studyvernment . Becausevernment . The maivernment . Misleadvernment . Sharingvernment .There have been only a few studies on crisis-related misinformation and mental health in Bangladesh. These include studies of misinformation concerning COVID-19 ,47,50,51RQ 1.How does perceived trust in sources of COVID-19 information vary by demographic/residency characteristics and frontline service provider status?RQ 2.To what extent does perceived trust in sources of COVID-19 information relate to COVID-19 stressors and mental health?RQ 3.Do COVID-19 stressors mediate associations between perceived trust in sources of COVID-19 information and mental health?The present study aims to fill the literature gaps identified above. We explore associations between perceived trust in sources of COVID-19 information, COVID-19 related stressors, and mental health in a low-income country (Bangladesh). Based on the available literature on media consumption during crises such as COVID-19, we pursued the following research questions (RQs):We used an online survey to collect the necessary data to answer our research questions. We started by developing a draft questionnaire and performing pilot surveys to refine the survey items. Next, we disseminated the final questionnaire see  between Eight sources were used to measure the perceived trust of information sources. These included government health agencies, international health agencies, healthcare personnel, social networks , online news portals, television, radio, and newspapers. For each information source, respondents were asked about their most trustworthy sources of COVID-19 information on a scale of 0  to 3 . We calculated the mean trust score for information sources and classified them into three groups as follows: (1) health media , (2) social media , and (3) traditional media . Survey responses were further divided into high levels of trust  or low levels of trust .A COVID-19 related stressors scale was modified from a 10-item SARS stressors assessment . For theMental health was assessed using the Generalized Anxiety Disorder (GAD-7) and Perceived Stress Scale (PSS-4). The GAD-7 is a validated scale to assess anxiety symptoms and generalized anxiety disorder . ParticiGender, age, education level, urbanicity, living status, and frontline service provider status were included as covariates. Gender was self-categorized as male/female. Age was split amongst two levels: ≤30 and >30 years old. Education status was defined by (1) school-level education, (2) college-level education, (3) at least some undergraduate education, (4) at least some graduate education, or (5) postgraduate education. School and college-level education were classified as having a low level of education. All others were classified as having a high level of education. Urbanicity was indicated by the respondents and included either currently living in an urban or a rural area. Living status was assessed by asking whether participants lived with family, non-family, or alone. This variable was later coded as a binary choice: living with family or not. Frontline service provider status was assessed by asking respondents whether they engaged directly with providing any emergency support during the COVID-19 pandemic. Working professionals, such as healthcare personnel, pharmaceutical professionals, banker, police, and government administrators, were included in this category.We addressed RQ 1 by counting the number of respondents in each demographic/residency group  and frontline provider status group  reporting high or low trust levels for each type of information source . Chi-square tests for categorical variables and Kruskal–Wallis tests for continuous variables were used to identify statistically significant differences in trust levels between groups.p < 0.05 was judged statistically significant.We addressed RQ 2 by conducting bivariate and multivariate regression analyses. We first calculated bivariate correlations between perceived trust in each information source , COVID-19 related stressors, and mental health outcomes . Next, we ran multiple linear regression models to determine how perceived trust in each information source predicted anxiety (Model 1) and stress (Model 2) while adjusting for demographic, residency characteristics, and frontline healthcare provider status. A two-tailed test with a significance level of Finally, we addressed RQ 3 by running a Preacher and Hayes mediation analysis to explore how COVID-19 related stressors explained the associations between perceived trust in information sources and mental health outcomes . Because2 = 2.24, p < 0.05) and traditional media  than respondents with high education levels . Larger proportions of urban residents showed high trust in health media , social media , and traditional media  than rural residents. Relatively more participants living with family reported higher trust in health media  and traditional media  than participants who did not live with family. No other differences in perceived trust in information sources were observed by demographics, residency, and frontline service provider status, p > 0.10.Perceived trust in information sources varied among demographics, residency, and frontline service provider status . Specifip < 0.001) and traditional media . COVID-19 related stressors were also positively correlated with anxiety . Perceived trust in all three information sources  was positively associated with anxiety  and negatively associated with perceived stress .p = 0.02) and stress (p = 0.03). Positive associations were also observed between perceived trust in traditional media and anxiety (p = 0.02). Perceived trust in traditional media was positively associated with anxiety (p = 0.03) but negatively associated with perceived stress (p = 0.04). Perceived trust in health media was not associated with either mental health outcome (p > 0.10).The direct, indirect, and total effects of perceived trust in information sources on mental health while considering COVID-19 related stressors as a putative mediator are displayed in The rapidly spreading behavior of COVID-19 has drastically altered the daily lives of people globally . As a reOur study investigated the role of perceived trust in COVID-19 related information sources in affecting mental health during the early stage of the pandemic in Bangladesh. COVID-19 related stressors were studied as putative mediators of the relationship between trust in that relationship. We first found important differences in perceived trust in sources of COVID-19 information across sociodemographic and residency characteristics (RQ 1). We then found that Bangladeshi citizens who trusted social and traditional media sources of COVID-19 information showed higher anxiety levels than citizens who trusted these sources less (RQ 2). Last, we found that COVID-19 related stressors partially explained associations between perceived trust in sources of COVID-19 information and anxiety (RQ 3).Many studies have already been conducted about the effects of trust in social media on people’s anxiety and stress levels. In nearly every case, anxiety and stress levels were associated with greater media consumption, and often with perceived trust ,15,61,62Interestingly, we found that trust in traditional media  was positively associated with COVID-19 related stress and anxiety . It is noteworthy that Bangladesh has recorded 30 television channels and 1191 daily newspapers that broadcast and print the country’s news . There aWe noted that perceived trust in COVID-19 information varied by education level, urbanicity, and living vs. not living with family (RQ 1). More specifically, relatively more highly educated people distrusted social media and traditional media than respondents with lower levels of education. This result is supported by previous findings that university graduates are less likely to believe in myths and information than less highly educated people . These rThe findings of this study have some theoretical and practical implications. Our study is the first of its kind in Bangladesh to examine associations between trust in information sources and mental health and test for a mediating role of COVID-19-related stress. Therefore, this study expands our understanding of trust and informational sources’ role on mental health during the COVID-19 pandemic. This study also introduces the novel finding that COVID-19 stressors play a role in affecting the impact of perceived trust in media on anxiety. Noteworthy is the finding that people are more vulnerable to anxiety in the presence of COVID-19 stressors and when holding low trust toward COVID-19 related information. The trustworthiness of unregulated and unverified COVID-19 information appears to interact with the presence of stressors  to impact mental health. Due to COVID-19 restrictions, many people were out of work and couldn’t participate in social activities. These restrictions provided ample opportunity to consume media content, which likely impacted their mental health. Thus, the findings of this study should encourage health authorities to properly educate social media users about the possible impact of mistrusted information during emergency periods. Our findings also suggest that if social media companies filter news and halt misinformation, these actions might reduce anxiety and stress among the global population.Additional recommendations can be based on these findings. Reliance on authenticated information sources, such as the Directorate General of Health Services (DGSH), Ministry of Health and Family Welfare , and the WHO should be encouraged. Frequent scrolling through COVID-19 news and social media forums should be limited. One of the best ways to prevent misleading information is to widely share accurate information . TherefoThis study has some limitations. First, our study design was cross-sectional, which was insufficient to explain the causal impacts of perceived trust or COVID-19 related stressors on mental health over time. Longitudinal studies are required to investigate these hypothesized relationships. Second, the online and self-reported surveys may have contained response biases. In particular, the study design could not reach residents without internet who may have experienced the pandemic and consumed media differently than our respondents. Third, we collected data in a limited time range during the early stage of the pandemic. As such, our findings may not translate to later periods of the COVID-19 pandemic. Fourth, the side-effects of vaccinations and the degree of awareness about the efficacy of the vaccine are subject to some debate amongst the public. Some support vaccinations, others refuse to get vaccinated, and in some contexts, large numbers of people are mandated to get vaccinated or face financial or employment ramifications. This study did not consider the effect of vaccinations on respondents’ mental health despite the potential for vaccination status/beliefs to confound the relationships we examined. Finally, the study did not consider whether family or close relatives tested positive for COVID-19, which could have had a substantial impact on mental health. Future research may incorporate additional insights concerning these topics by including covariates, such as vaccination coverage, testing positive with COVID-19, comorbidities, long-term illness, and pre-history mental illness. Future research could also focus on associations between trust in various media platforms and mental health during other types of emergencies, such as future pandemics, severe weather events, and other natural disasters. Evidence has shown that limited social support triggers negative mental health consequences. Thus, social support may have moderated the influence on mental health and this possibility could be addressed in future studies as well.In a nationwide cross-sectional study of Bangladeshi citizens, we found that trust in sources of COVID-19 information was higher amongst better-educated residents, urban dwellers, and those living with family. Greater trust in COVID-19 information sources was related to more stress and anxiety. COVID-19 related stressors mediated the relationships between perceived trust in information sources and anxiety. Based on these findings, attention should be paid to media consumption, its creditability, and mental health outcomes of Bangladeshi residents. Responsible authorities should broadcast authentic news and misinformation and rumors on social media should be minimized.