For many decades, there have been numerous reported cases of falsified liquid medical products, including vaccine and insulin preparations worldwide, but to date, there has been a lack of affordable and accessible analytical methods for biological medicines and vaccine authenticity testing. A conventional clinical chemistry analyser (Abbott Architect c16000) was used to determine the concentrations of analytes in genuine liquid biological products (vaccines and insulin) and falsified vaccine surrogates. Eight analytes were measured for each sample: sodium, potassium, chloride, calcium, magnesium, phosphate, glucose and protein. Each genuine liquid product had unique concentrations of analytes when tested using the eight methods applied, allowing clear differentiation from the falsified surrogates. In a blinded study, reproducibility was significantly high when the samples were run intra- and inter-batch up to 9 times over 9 different days, and it was possible to identify most of the samples by analyte presence alone. Imprecision was < 1.0 CV% for ion-selective electrode methods and typically < 5 CV% for spectrophotometric methods. A decision tree was created which was able to identify all samples. We demonstrate for the first time that a conventional clinical chemistry analyser provides a low-cost method to accurately differentiate genuine products from falsified surrogate liquid medicines and vaccines. This novel method has the potential to be used globally due to widespread use of clinical chemistry analysers in hospitals across the world, including in low- and middle-income countries where many cases of falsified medicines have been identified.

Soluble Fms-like tyrosine kinase 1 (sFlt-1), a protein secreted by the placenta, plays a central role in the pathogenesis of preeclampsia-a life-threatening pregnancy complication for which no disease-specific treatment currently exists. We developed a strategy to selectively deplete circulating sFlt-1 and then conducted a single-arm, open-label trial to reduce circulating sFlt-1 in women with very preterm preeclampsia. The primary endpoints were safety and tolerability. Extracorporeal apheresis with an adsorber containing high-affinity IgG1 antibodies against sFlt-1 resulted in an approximately 50% reduction of circulating sFlt-1 levels in pregnant baboons. In women with preterm preeclampsia treated with single ascending doses (phase A, n = 7, preapheresis, mean ± s.d., sFlt-1: 15,120 ± 4,484 pg ml-1), maternal and fetal vital signs and umbilical artery pulsatility indices remained stable when comparing measures before, during and after apheresis. In women with very preterm preeclampsia treated with multiple doses (phase B, n = 9, median gestational age 30.3 (interquartile range, 29.3-30.9) weeks, systolic and diastolic blood pressures 146 ± 10 and 92 ± 5 mmHg, respectively, and preapheresis circulating sFlt-1 levels 11,960 ± 3,056 pg ml-1), each apheresis reduced sFlt-1 levels by 16.7 ± 7.6% and mean arterial pressures by 4.1 ± 7.8 mmHg. Reductions in mean arterial pressures after apheresis strongly correlated with reductions in circulating sFlt-1 (R = 0.63, Spearman's correlation). Pregnancy continued from admission for a median of 10 (range, 3-19) days. Compared to antenatal estimated birth weights, neonatal birth weights generally remained stable or increased among those with the longest extensions. Treatment-related adverse events included mild hypocalcemia (n = 3), skin hemorrhage at the puncture site (n = 1) and false labor (n = 1). Selective removal of sFlt-1 by apheresis appeared to be safe and well tolerated in women with very preterm preeclampsia. Controlled trials are needed to confirm the additional safety and efficacy of this approach. ClinicalTrials.gov registration: NCT02923206 .

Introduction:The measurement of the angiogenic biomarkers placental growth factor (PlGF) and soluble fms-like tyrosine kinase-1 (sFlt-1) are increasingly used to support the prediction and diagnosis of preeclampsia (PE) in routine clinical practice. There are an increasing number of methods available for the analysis of these markers but data showing their analytical comparability is limited. Materials and methods:The assays for PlGF and sFlt-1 from Revvity were evaluated and compared against Roche methods that are used in current clinical practice in Oxford. Imprecision and paired analytical comparisons studies were undertaken and data evaluated for numeric agreement and concordance relative to manufacturer recommended rule-in and rule-out thresholds for PE. Results:Imprecision estimates for the Revvity PlGF and sFlt-1 methods calculated from quality control material analysed during the evaluation were between 3.2 and 9.0 CV%. Revvity method precision profiles derived from 581 clinical specimens analysed in duplicate had a median CV% for PlGF of 1.8%, IQR 2.3% and for sFlt-1 a median CV% of 1.1%, IQR 1.5%. Comparison against the Roche PlGF and sFlt-1 methods in 437 clinical specimens showed an overall Passing-Bablok regression relationship for PlGF of y = -23.4 + 0.73x (r = 0.983) and for sFlt-1, y = -87.7 + 0.40x (r = 0.971). However, there was statistically significant (p < 0.0001) concentration dependant relative bias for both methods and the calculated ratio. Concordance of the sFlt-1:PlGF ratio relative to manufacturer specific rule-in and rule-out thresholds was 95.2%. Discussion:The Revvity methods for PlGF and sFlt-1 are precise and correlate with the Roche methods. However, numeric agreement precludes result interchangeability and the use of common rule-in and rule-out thresholds. The manufacturer specific thresholds should be applied in clinical practice. Further work is required to understand how method differences impact clinical outcomes and their causes.

AIMS:To assess the impact of adding clinical comments to reports of thyroid function testing in patients treated for hypothyroidism. METHODS:We compared thyroid function test results in primary care patients being treated for hypothyroidism from January 2016 to August 2023 at two NHS Trusts with similar demographics and using the same instruments, but with different interpretative comment policies. One laboratory, Buckinghamshire Health Trust (Bucks), adds interpretative comments, whereas the other, Oxford University Hospitals (Oxford), does not. We used two outcome measures: the percentage of patients with thyroid-stimulating hormone (TSH) within the reference interval on repeat testing and the timing of repeat TSH testing samples, according to the National Institute for Health and Care Excellence guidance (NG145). RESULTS:We identified 18 242 and 31 655 hypothyroid patients (9.0% and 7.7% of the population tested) in Bucks and Oxford, with a total of 121 961 and 247 639 tests over the evaluation period, respectively. The proportion of TSH results within the reference interval (83.4% in Bucks, 83.9% in Oxford) was similar in both Trusts, as was TSH concentration (median TSH concentration 1.60 (IQR 0.78-2.82) mU/L in Bucks, 1.68 (IQR 0.97-2.76) in Oxford). The interval between tests was shorter in Oxford, but differed significantly from NG145 in both Trusts. Differences were statistically significant for both outcome measures, but of questionable clinical significance. CONCLUSIONS:Adding interpretative comments to results of thyroid function tests does not appear to affect the distribution of TSH concentrations in primary care patients on thyroxine replacement or the intervals between tests in a clinically meaningful way.

Abstract L-carnitine is critical for metabolism of some fatty acids therefore different carnitine species may play a role in metabolic dysfunction-associated steatotic liver disease (MASLD) pathogenesis. Until recently, hepatic acetylcarnitine could not be quantified non-invasively. Now new technical developments in magnetic resonance spectroscopy (MRS) enable quantification of hepatic acetylcarnitine content in vivo. The aim of this study was to evaluate potential metabolic changes in healthy volunteers and patients with different stages of MASLD before and after a single injection of L-carnitine. This prospective study recruited 10 healthy volunteers and 17 patients (11 with low-risk MASLD, 6 with high-risk MASLD) who underwent cardiac and hepatic MRI and MRS for hepatic acetylcarnitine quantification at baseline and 2 h after injecting 50 mg/kg body weight L-carnitine. Serum samples were obtained for biomarker measurement. A single injection of L-carnitine significantly elevated hepatic acetylcarnitine levels in healthy volunteers (Δ ACC = 0.172, p = 0.038) and low-risk MASLD (Δ ACC = 1.677, p = 0.043), but not in high-risk MASLD (Δ ACC =-0.144, p = 0.397). However, high-risk MASLD showed elevated hepatic acetylcarnitine at baseline (Δ ACC = 0.58, p = 0.007), whereas variability in low-risk MASLD masked differences vs. healthy volunteers. At baseline, serum medium-chain carnitine species were higher in the high-risk MASLD group (0.88 ± 0.28µmol/L, p = 0.002) and low-risk MASLD group (0.77 ± 0.49µmol/L, p = 0.03) compared to healthy volunteers (0.36 ± 0.23µmol/L). We showed for the first time that acetylcarnitine can be evaluated in vivo in patients with different stages of MASLD. The divergent hepatic responses to L-carnitine suggest stage-dependent differences in hepatic metabolic response that may reflect disease severity, potentially arising from differences in metabolic flexibility, carnitine transport, mitochondrial function, or baseline saturation of hepatic acetylcarnitine. Further studies are needed to confirm whether hepatic or circulating acetylcarnitine levels could serve as early biomarkers of MASLD progression.

A faecal immunochemical test (FIT) result ≥ 10 µg/g is recommended in the UK to triage patients with symptoms of colorectal cancer (CRC) in primary care for urgent cancer investigation. The COLOFIT model combining FIT results with demographics and blood tests was developed to reduce the proportion of people referred without CRC. This study aims to externally validate the COLOFIT using data from Oxford University Hospitals (OUH). FITs requested by GPs between January 2017 and February 2024 were extracted from the OUH Clinical Data warehouse. Adults with COLOFIT predictors and 180-day follow-up for CRC were included. External validation of the COLOFIT equation was conducted overall and for six independent time periods. Risk score thresholds where the model captured the same number of cancers as FIT ≥ 10 µg/g were estimated to understand the number of urgent referrals avoided. A total of 51,477 individuals (659 CRC) were included; 6194 (12

OBJECTIVE:Primary aldosteronism (PA) is the commonest secondary cause of hypertension but case-detection remains a challenge. Screening is usually performed in secondary care using an aldosterone:renin ratio (ARR) measurement. Here, we describe the outcomes of screening in primary care, in Oxfordshire, UK. DESIGN:Retrospective observational study. PATIENTS:Adults screened for PA in primary care services in Oxford between 2008 and 2022. MEASUREMENTS:ARR test results in primary care and outcomes of secondary care evaluation (ARR, saline infusion test, final diagnosis). Primary care and secondary care ARR tests were compared for correlation, concordance and performance in predicting PA. RESULTS:Among 2915 adults screened in primary care, 455 were referred to secondary care and 107 (3.7% of total population screened) were diagnosed with PA. Primary care ARR showed strong correlation with secondary care ARR (r = 0.841, p < 0.001). Area under the ROC curve to predict PA was 0.81 (95% CI 0.77-0.86) for primary care ARR testing. Primary care ARR cut-off of ≥ 30 pmol/mU showed comparable sensitivity (91.7% vs 92.1%, p = 0.467) to and modest concordance (Kappa 0.583, p < 0.001) with secondary care ARR. Use of beta-blockers were associated with higher risk of false positive test result (OR 3.5, 95% CI 1.1-12.0, p = 0.042). CONCLUSIONS:Screening for PA in primary care with ARR is feasible with modest concordance and comparable sensitivity to secondary care testing. Simple referral criteria and raising awareness among primary care colleagues could ensure appropriate referral to secondary care.

Maintaining cold-chain integrity is vital for vaccines to ensure that they remain within the recommended temperature limits during routine storage and transportation. This ensures vaccine stability, efficacy, and avoids degradation. Here, we propose rapid and low-cost tests based on simple glucose assays to detect heat-exposed degraded sucrose-containing vaccines through sucrose's inherent gradual conversion to glucose when exposed to elevated temperatures. Bioluminescent and colorimetric assays and a clinical biochemical analyser for urine samples could successfully determine effects of heat exposure on vaccines by detecting a significant increase in glucose levels. We show that this increase in glucose also correlates with the loss of vaccine potency. When vaccines were incubated at 37 and 45 °C, the bioluminescent assay was able to detect an increase in glucose levels from 12 h of heat exposure. The biochemical analyser could successfully detect increased glucose levels in a COVID-19 vaccine which had been exposed to 37 and 45 °C. Most importantly, the colorimetric assay has the advantage of producing a colour change visually upon simply mixing the vaccine with a reagent without the need for a plate reader or any other sophisticated devices. To our knowledge, this is the first simple, rapid and device-free test of its kind to detect heat-exposed substandard vaccines, making it a potential test for deploying at key points in the supply chain in warm and hot countries to check the integrity of vaccine cold-chain. Although this test does not replace the more definitive lot release assays such as potency assays, it could initially be used as a rapid and low-cost test to identify substandard sucrose-containing vaccines within supply chains, in support of WHO's Prevent, Detect, and Respond strategy.

This chapter looks at fluid and electrolyte balance from the viewpoint of the clinical biochemistry laboratory; it considers the major causes of each abnormality that may be encountered and provides guidance on the accurate reporting of these parameters. The plasma electrolytes sodium and potassium are the most frequently requested investigations in most clinical biochemistry laboratories. Ion-selective electrodes (ISEs) are the main analytical technique used for analysis of sodium and potassium. Two distinct types of ISE, direct and indirect, are used to measure electrolyte concentrations and these may produce different results when used for specimens in which the total protein and/or lipid contents are increased. Poor collection and handling of clinical samples can adversely affect plasma potassium test results. Ultimately, serious disturbances to plasma electrolyte concentrations can be life threatening and rapid communication of abnormal test results by laboratory staff to clinical teams is essential.

Kidney transplantation is the preferred treatment for end-stage renal disease and is limited by donor organ availability. Normothermic Machine Perfusion (NMP) might facilitate safe transplantation of marginal organs. NKP1 is a single centre, phase 1, 36-patient, three-stage cohort study investigating the safety and feasibility of up to 24 hours of renal NMP prior to transplantation. 30-day graft survival (primary endpoint) was 100%. Secondary objectives were assessment of the effect of NMP on post-transplant clinical outcomes and ischaemia-reperfusion injury, identification of predictive biomarkers, and characterisation of the performance of the preservation system. Clinical outcomes were comparable to a matched control cohort with 12-month estimated glomerular filtration rate (eGFR) 46.3 vs 49.5 mL/min/1.73m2 (p = 0.44) despite much longer total preservation times (15.7 vs 8.9 hours controls, p < 0.0001). We saw strong correlations between biomarkers measured ex-situ and post-transplant outcomes, including graft function at one year (correlation between GST-Pi delta and 12-month eGFR, R = 0.54, p = 0.001). Renal NMP is useful for optimising logistics and as an organ assessment technique, and has potential to expand the donor pool. Trial registration number: ISRCTN13292277.

BACKGROUND: The ratio of soluble fms-like tyrosine kinase 1 to placental growth factor is a useful biomarker for preeclampsia. Since it is a measure of placental dysfunction, it could also be a predictor of clinical deterioration and fetal tolerance to intrapartum stress. OBJECTIVE: We tested the hypothesis that soluble fms-like tyrosine kinase 1 to placental growth factor ratio predicts time to delivery. Secondary objectives were to examine associations between the soluble fmslike tyrosine kinase 1 to placental growth factor ratio and mode of birth, fetal distress, need for labor induction, and birthweight z score. STUDY DESIGN: Secondary analysis of the INSPIRE trial, a randomized interventional study on prediction of preeclampsia/eclampsia in which women with suspected preeclampsia were recruited and their blood soluble fms-like tyrosine kinase 1 to placental growth factor ratio was assessed. We stratified participants into 3 groups according to the ratio result: category 1 (soluble fms-like tyrosine kinase 1 to placental growth factor <38); category 2 (soluble fms-like tyrosine kinase 1 to placental growth factor >38 and <85); and category 3 (soluble fms-like tyrosine kinase 1 to placental growth factor >85). We modeled time from soluble fms-like tyrosine kinase 1 to placental growth factor determination to delivery using Kaplan-Meier curves and compared the 3 ratio categories adjusting for gestational age at soluble fms-like tyrosine kinase 1 to placental growth factor determination and trial arm with Cox regression. The association between ratio category and mode of delivery, induction of labor, and fetal distress was assessed using a multivariable logistic regression adjusting for gestational age at sampling and trial arm. The association between birthweight z score and soluble fms-like tyrosine kinase 1 to placental growth factor ratio was evaluated using multiple linear regression. Subgroup analysis was conducted in women with no preeclampsia and spontaneous onset of labor; women with preeclampsia; and participants in the nonreveal arm. RESULTS: Higher ratio categories were associated with a shorter latency from soluble fms-like tyrosine kinase 1 to placental growth factor determination to delivery (37 vs 13 vs 10 days for ratios categories 1-3 respectively), hazards ratio for category 3 ratio of 5.64 (95% confidence interval 4.06-7.84, P<.001). A soluble fms-like tyrosine kinase 1 to placental growth factor ratio >85 had specificity of 92.7% (95% confidence interval 89.0%-95.1%) and sensitivity of 54.72% (95% confidence interval, 41.3-69.5) for prediction of preeclampsia indicated delivery within 2 weeks. A ratio category 3 was also associated with decreased odds of spontaneous vaginal delivery (Odds ratio [OR] 0.47, 95% confidence interval 0.25-0.89); an almost 6-fold increased risk of emergency cesarean section (OR 5.89, 95% confidence interval 3.05-11.21); and a 2-fold increased risk for intrapartum fetal distress requiring operative delivery or cesarean section (OR 3.04, 95% confidence interval 1.53-6.05) when compared to patients with ratios <38. Higher ratio categories were also associated with higher odds of induction of labor when compared to ratios category 1 (category 2, OR 2.20, 95% confidence interval 1.02-4.76; category 3, OR 6.0, 95% confidence interval 2.01-17.93); and lower median birthweight z score. Within subgroups of women a) without preeclampsia and with spontaneous onset of labor and b) women with preeclampsia, the log ratio was significantly higher in patients requiring intervention for fetal distress or failure to progress compared to those who delivered vaginaly without intervention. In the subset of women with no preeclampsia and spontaneous onset of labor, those who required intervention for fetal distress or failure to progress had a significantly higher log ratio than those who delivered vaginaly without needing intervention. CONCLUSION: The soluble fms-like tyrosine kinase 1 to placental growth factor ratio might be helpful in risk stratification of patients who present with suspected preeclampsia regarding clinical deterioration, intrapartum fetal distress, and mode of birth (including the need for intervention in labor).

We describe three cases of critical acute myositis with myocarditis occurring within 22 days of each other at a single institution, all within 1 month of receiving the initial cycle of the anti-PD-1 drug pembrolizumab. Analysis of T cell receptor repertoires from peripheral blood and tissues revealed a high degree of clonal expansion and public clones between cases, with several T cell clones expanded within the skeletal muscle putatively recognizing viral epitopes. All patients had recently received a COVID-19 mRNA booster vaccine prior to treatment and were positive for SARS-CoV2 Spike antibody. In conclusion, we report a series of unusually severe myositis and myocarditis following PD-1 blockade and the COVID-19 mRNA vaccination.

Background Faecal immunochemical testing (FIT) is recommended by the National Institute for Health and Care Excellence to triage symptomatic primary care patients who have unexplained symptoms but do not meet the criteria for a suspected lower gastrointestinal cancer pathway. During the COVID-19 pandemic, FIT was used to triage patients referred with urgent 2-week wait (2ww) cancer referrals instead of a direct-to-test strategy. FIT-negative patients were assessed and safety netted in a FIT negative clinic. Methods We reviewed case notes for 622 patients referred on a 2ww pathway and seen in a FIT negative clinic between June 2020 and April 2021 in a tertiary care hospital. We collected information on demographics, indication for referral, dates for referral, clinic visit, investigations and long-term outcomes. Results The average age of the patients was 71.5 years with 54% female, and a median follow-up of 2.5 years. Indications for referrals included: anaemia (11%), iron deficiency (24%), weight loss (9%), bleeding per rectum (5%) and change in bowel habits (61%). Of the cases, 28% (95% CI 24% to 31%) had endoscopic (15%, 95% CI 12% to 18%) and/or radiological (20%, 95% CI 17% to 23%) investigations requested after clinic review, and among those investigated, malignancy rate was 1.7%, with rectosigmoid neuroendocrine tumour, oesophageal cancer and lung adenocarcinoma. Conclusion A FIT negative clinic provides a safety net for patients with unexplained symptoms but low risk of colorectal cancer. These real-world data demonstrate significantly reduced demand on endoscopy and radiology services for FIT-negative patients referred via the 2ww pathway.

Abstract Objective This study evaluates the predictive value of copeptin for syndrome of inappropriate antidiuresis (SIAD) postpituitary transsphenoidal surgery (TSS). Design Data from 133 consecutive patients undergoing TSS (November 2017–October 2022) at Oxford University Hospitals NHS trust are presented in this retrospective study. Methods Logistic regression (LR) and receiver operating characteristic (ROC) curves were performed to evaluate the diagnostic utility of copeptin. The Mann–Whitney U test was used to compare copeptin levels between the SIAD and no SIAD groups. Results Fourteen patients (10.8%) developed SIAD. Copeptin was available in 121, 53 and 87 patients for Days 1, 241 and 8 post‐TSS, respectively. LR for Day 1 copeptin to predict SIAD gave an odds ratio (OR) of 1.0 (95%CI 42 0.84–1.20, p = .99), area under‐ROC curve (AUC) was 0.49; Day 2 copeptin OR was 0.65 (95%CI 0.39–1.19, 43 p = .77), AUC was 0.57 LR for Day 1 sodium to predict SIAD gave an odds ratio (OR) of 1.0 (95%CI 0.85–1.21, p = .99), AUC was 0.50. Conclusions In conclusion, our data provide no evidence for copeptin as a predictive marker for post‐TSS SIAD.

Kidney transplantation is the preferred treatment for end-stage renal disease and is limited by donor organ availability. Normothermic Machine Perfusion (NMP) might facilitate safe transplantation of marginal organs. Previous clinical implementations have been limited to short perfusions. NKP1 was a single centre, phase 1, 36-patient, three-stage cohort study investigating the safety and feasibility of up to 24 hours of renal NMP prior to transplantation. We observed a 30-day graft survival of 100%, with comparable outcomes to a matched control cohort (12-month estimated glomerular filtration rate (eGFR) 46.3 vs 49.5mL/min/1.73m2, p=0.44) despite much longer total preservation times (15.7 vs 8.9 hours controls, p <0.0001). We saw strong correlations between biomarkers measured ex-situ and post-transplant outcomes, including graft function at one year (correlation between GST-Pi delta and 12-month eGFR, R=0.54, p=0.001). Renal NMP is useful for optimising logistics and as an organ assessment technique, and has potential to expand the donor pool. Trial registration number: ISRCTN13292277.

Abstract Background An outbreak of acute severe hepatitis of unknown aetiology (AS-Hep-UA) in children during 2022 was subsequently linked to infections with adenovirus-associated virus 2 and other ‘helper viruses’, including human adenovirus. It is possible that evidence of such an outbreak could be identified at a population level based on routine data captured by electronic health records (EHR). Methods We used anonymised EHR to collate retrospective data for all emergency presentations to Oxford University Hospitals NHS Foundation Trust in the UK, between 2016–2022, for all ages from 18 months and older. We investigated clinical characteristics and temporal distribution of presentations of acute hepatitis and of adenovirus infections based on laboratory data and clinical coding. We relaxed the stringent case definition adopted during the AS-Hep-UA to identify all cases of acute hepatitis with unknown aetiology (termed AHUA). We compared events within the outbreak period (defined as 1st Oct 2021—31 Aug 2022) to the rest of our study period. Results Over the study period, there were 903,433 acute presentations overall, of which 391 (0.04%) were classified as AHUA. AHUA episodes had significantly higher critical care admission rates (p < 0.0001, OR = 41.7, 95% CI:26.3–65.0) and longer inpatient admissions (p < 0.0001) compared with the rest of the patient population. During the outbreak period, significantly more adults (≥ 16 years) were diagnosed with AHUA (p < 0.0001, OR = 3.01, 95% CI: 2.20–4.12), and there were significantly more human adenovirus (HadV) infections in children (p < 0.001, OR = 1.78, 95% CI:1.27–2.47). There were also more HAdV tests performed during the outbreak (p < 0.0001, OR = 1.27, 95% CI:1.17–1.37). Among 3,707 individuals who were tested for HAdV, 179 (4.8%) were positive. However, there was no evidence of more acute hepatitis or increased severity of illness in HadV-positive compared to negative cases. Conclusions Our results highlight an increase in AHUA in adults coinciding with the period of the outbreak in children, but not linked to documented HAdV infection. Tracking changes in routinely collected clinical data through EHR could be used to support outbreak surveillance.

ABSTRACT Background An outbreak of severe acute hepatitis of unknown aetiology (AS-Hep-UA) in children during 2022 has subsequently been linked to infections by adenovirus-associated virus 2 (AAV2) and other ‘helper viruses’, including human adenovirus (HAdV). Aim We investigated clinical characteristics and temporal distribution of acute hepatitis with unknown aetiology (AHUA) and of HAdV infections in Oxfordshire, UK population between 2016-2022. Methods We used anonymised electronic health records (EHR) to collate retrospective data for presentations of AHUA and/or HAdV infection between 2016-2022. We reviewed records of >900,000 acute presentations to emergency care at Oxford University Hospitals NHS Foundation Trust (OUH; UK) and performed a descriptive analysis of case numbers and clinical characteristics. Results Over the full study period, patients coded as AHUA had significantly higher critical care admission rates (p<0.0001, OR=41.7, 95% CI:26.3-65.0) and longer inpatient admissions (p<0.0001) compared with the rest of the patient population. Comparing events within the outbreak period (1st Oct 2021 - 31 Aug 2022), to those occurring outside this period, significantly more adults were diagnosed with AHUA (p<0.0001, OR=3.01, 95% CI: 2.20-4.12), and there were significantly more HAdV infections in children (p<0.001, OR=1.78, 95% CI:1.27-2.47). There were also more HAdV tests administered during the outbreak (p<0.0001, OR=1.27, 95% CI:1.17-1.37). There was no evidence of more acute hepatitis or increased severity of illness among patients who tested HAdV-positive compared to those testing HAdV-negative. Conclusion Our results highlight an increase in the number of AHUA in adults coinciding with the reported AS-Hep-UA outbreak in children, but not linked to documented HAdV infection.

Exclusion criteria for reference studies: Context is key We read with interest the response to our letter on pregnancy-specific reference intervals and the significance of body mass index (BMI). We welcome the opportunity to address some of the key points. ‘The values used in determining reference intervals are always selected from disease-free subjects’. The IFCC’s series on defining reference intervals recommends that ‘health is not a well-defined condition and has come to be regarded as relative’, and ‘...in selecting reference individuals, the criteria of health applied are dictated by the aim of the laboratory investigation. Thus, [reference intervals] may not always be healthy subjects’. If a state of absolute health does not exist, a transparent approach is required for risk stratification in reference studies. Such an approach may include the use of risk factors as proxies. For example, a reference study for a novel cardiac marker should probably exclude those with a personal history of myocardial infarction, but it may also be reasonable to exclude those with a higher risk of unreported or undiagnosed disease, like obesity. In contrast, while not strictly ‘healthy’, such a reference population may include individuals with asthma or arthritis. Context is key. ‘Obesity is a biological disadvantage to the person and thus fulfils the definition of a disease’. Older age, female sex and obesity predispose to the development of breast cancer, but neither of these is a disease. While being mindful of the aforementioned potential utility of proxy markers, caution should be taken to differentiate between diseases and their risk factors. ‘Just because more than 50% of women in Britain and Ireland are overweight or obese is not a reason to pretend all are “normal”. Interpreting tests in the context of BMI is indeed important if the interpretation recognises that obesity is an illness.’ Whether one chooses to classify obesity as a disease or not, the fact is that women with a higher BMI constitute a significant proportion of the pregnant population, and they need reliable diagnostic tests. If the reference interval for an analyte differs by BMI there are two options: 1) create ‘high’ and ‘low risk’ alternatives a priori or 2) recruit inclusively and partition properly. Ultimately, the desired outcome is the same: ensuring that clinical tests are interpreted safely and appropriately in the context of the target population. We strongly believe that exclusion based on BMI should be the exception, not the rule.