Band 59, Heft 5

This review summarizes and critically evaluates the published approaches and recent trends in sample pre-treatment, as well as both separation and non-separation techniques used for the determination of uric acid (UA) in saliva. UA is the final product of purine nucleotide catabolism in humans. UA concentrations in biological fluids such as serum, plasma, and urine represent an important biomarker of diseases including gout, hyperuricemia, or disorders associated with oxidative stress. Previous studies reported correlation between UA concentrations detected in saliva and in the blood. The interest in UA has been increasing during the past 20 years from a single publication in 2000 to 34 papers in 2019 according to MEDLINE search using term “uric acid in saliva”. The evaluation of salivary UA levels can contribute to non-invasive diagnosis of many serious diseases. Increased salivary UA concentration is associated with cancer, HIV, gout, and hypertension. In contrast, low UA levels are associated with Alzheimer disease, progression of multiple sclerosis, and mild cognitive impairment.

Basophils (basophilic granulocytes) are the least abundant cells in blood. Nowadays, basophils are included in the complete blood count performed by hematology analyzers and therefore reported in practically all patients in whom hematologic investigations are requested. However, hematology analyzers are not reliable enough to report clinically useful results. This is due to a combination of very high analytical imprecision and poor specificity, because the chemical and physical methods used for basophil counting in hematology analyzers are ill-defined and thus basophils are not well recognized by the analyzers. As a result, false basophil counts are quite common. In view of increasing analytical performance demands, hematology laboratories should stop reporting basophil counts produced by hematology analyzers. Suggestions for alternative pathways are presented for those situations where basophils are of clinical relevance.

Starting from the discussion topics triggered by Hoffmann about the past and current basophil counting, a broader view of the role and future of laboratory hematology, passing through some general considerations concerning the idea of laboratory medicine in the healthcare pathway between technology and professionalism, is here provided.

Home pregnancy tests (HPTs) available in Europe include accuracy and other performance claims listed on their packaging. Due to the lack of guidance on the standardisation of such products, it is often difficult to replicate these claims when tested on a clinical sample, whether in a laboratory setting or by lay users. The In Vitro Diagnostic Regulation is a set of requirements that mandate comprehensive validation data on human pregnancy tests and other in vitro devices. It is due to replace the current European Directive (98/79/EC) and fully implemented in Europe by 2022. In June 2019, a panel of seven experts convened to discuss the validation studies required to provide the information needed to meet the new regulation for HPTs in Europe and proposed 15 recommendations for best practice. Defining best practice at all stages of validation of these important tests may ensure that tests marketed in Europe are fit for purpose, enabling lay users to be confident of the high quality of the HPT results they obtain. The panelists believe that the recommendations proposed here for the validation of HPTs may constructively contribute to improved standardisation of validation procedures in Europe.

Objectives The utilization of reliable quality indicators (QIs) proven to be suitable for monitoring and improvement tools is one of the best choices to minimize of the risk of errors in all laboratory processes called as total testing process (TTP). In 2008, a Working Group “Laboratory Errors and Patient Safety” (WG-LEPS) established by International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) developed the Model of Quality Indicators (MQI) complying with requirements of the ISO 15189:2012 standard for laboratory accreditation. They have also been dealing with harmonizing the QIs in most laboratories worldwide since then. The present study was set out to investigate the frequency of using IFCC WG-LEPS’ pre-QIs by Turkish laboratories and to assess the conformity of them, by taking into account Turkey’s conditions. Methods A survey consisting nine questions was applied in 81 laboratories using SurveyMonkey. Results According to the survey results, most of the laboratories reported they have used pre-QIs in the quality standards of health prepared by Turkish Ministry of Health (MOH). A part of IFCC WG-LEPS’ pre-QIs were being utilized by more than 80% of the laboratories, the rest of which only used by 10% of laboratories. Conclusions The majority of the medical laboratories have been using the pre-QIs included in the guidelines of Quality Standards prepared by the MOH. The pre-QIs are partially compatible with IFCC WG-LEPS’ pre-QIs. The definitions of IFCC WG-LEPS’ pre-QIs may also be revised to make them more clear and understandable by IFCC WG-LEPS. The insufficiency of Health Information Management Systems (HIMS) limits the use of pre-QIs proposed by IFCC WG-LEPS. Finally, the education of relevant personnel about the use of HIMS and pre-QIs is very crucial to harmonize and to extend the use of IFCC WG-LEPS’ pre-QIs in Turkish medical biochemistry laboratories.

Objectives In spite of the introduction of automated systems for urinary sediment analysis, microscopy examination remains the gold standard, and it is more than ever important to perform it with a good and reliable quality. External Quality Assessment (EQA) programs on urinary sediment are rare. The present paper provides an analysis of results from 2001 to date of the EQA Italian program which involves today 230 laboratories. Methods The program includes four surveys per year. Participants are asked the identification and clinical associations of urinary sediment particles, shown as phase contrast microscopy images in the website of the Center of Biomedical Research (CRB) (2 surveys), and the diagnosis of clinical cases presented by both images and a short clinical history (2 surveys). The results of each survey are then scored and commented. In 20 years, 298 images were presented: 90 cells (9 types), 23 lipids (5 types), 87 casts (21 types), 53 crystals (14 types), 22 microorganisms (5 types), and 23 contaminants (9 types). Moreover, 27 clinical cases, covering a wide spectrum of conditions with different degrees of complexity, were presented to participants. Results Identification: among urinary particle categories, the correct identification rate (obtained for each particle from the sum of correct + partially correct answers) was very high for micro-organisms (mean ± SD: 96.2 ± 3.5%), high for lipids (88.0 ± 11.8%) and crystals (87.0 ± 16.5%) followed, in decreasing order, by cells (82.1 ± 15.9%), casts (81.8 ± 14.8%), and contaminants (76.7 ± 22.1%). Clinical associations (n=67): the rate of correct answers was 93.5 ± 5.7% ranging from 75.0 to 100% for all but one clinical association (i.e., acute glomerulonephritis: 55.4%). Clinical cases: throughout surveys, due to the overall rate of particle misidentification, only 59.8 ± 17.1%, (range 32.5–88.7%) of participants achieved access to clinical diagnosis. Of these, 88.7 ± 10.6% (range 59.9–99.3%) were able to indicate the correct diagnosis. Conclusions Our program can be used as a tool to improve the identification of urine particles and the knowledge of their clinical meaning and to encourage specialists of laboratory medicine to correlate urinary findings with other laboratory data and the clinical history, an aspect that improves the value of the day by day work.

Objectives Low-density lipoprotein cholesterol (LDL-C) is the main laboratory parameter used for the management of cardiovascular disease. The aim of this study was to compare measured LDL-C with LDL-C as calculated by the Friedewald, Martin/Hopkins, Vujovic, and Sampson formulas with regard to triglyceride (TG), LDL-C and non-high-density lipoprotein cholesterol (non-HDL-C)/TG ratio. Methods The 1,209 calculated LDL-C results were compared with LDL-C measured using ultracentrifugation-precipitation (first study) and direct (second study) methods. The Passing-Bablok regression was applied to compare the methods. The percentage difference between calculated and measured LDL-C (total error) and the number of results exceeding the total error goal of 12% were established. Results There was good correlation between the measurement and calculation methods (r 0.962–0.985). The median total error ranged from −2.7%/+1.4% (first/second study) for Vujovic formula to −6.7%/−4.3% for Friedewald formula. The numbers of underestimated results exceeding the total error goal of 12% were 67 (Vujovic), 134 (Martin/Hopkins), 157 (Samspon), and 239 (Friedewald). Less than 7% of those results were obtained for samples with TG >4.5 mmol/L. From 57% (Martin/Hopkins) to 81% (Vujovic) of underestimated results were obtained for samples with a non-HDL-C/TG ratio of <2.4. Conclusions The Martin/Hopkins, Vujovic and Sampson formulas appear to be more accurate than the Friedewald formula. To minimize the number of significantly underestimated LDL-C results, we propose the implementation of risk categories according to non-HDL-C/TG ratio and suggest that for samples with a non-HDL-C/TG ratio of <1.2, the LDL-C level should not be calculated but measured independently from TG level.

Objectives The value of the serum protein gap (PG, difference between total protein and albumin) in the detection of hyper- or hypogammaglobulinemia is not well established. We assessed the performance of PG for the detection of hyper- or hypogammaglobulinemia in a large sample of patients. Methods We reviewed all paired measurements of serum total protein, albumin, quantitative immunoglobulins, and serum protein electrophoresis tested between March 2014 and June 2017 at the Eastern Ontario Regional Laboratory Association. Sensitivity, specificity, positive predictive value, negative predictive value and likelihood ratios of PG at thresholds between 18 and 44 g/L for the detection of hyper- and hypogammaglobulinemia were assessed. Results There were 19,575 and 5,426 simultaneous paired data points to assess hyper- and hypogammaglobulinemia identified by serum protein electrophoresis (SPE) and nephelometry, respectively. The mean PG was 36.3 g/L (SD 8.6). The prevalence of hypergammaglobulinemia (>16 g/L by SPE) and hypogammaglobulinemia (IgG <7 g/L) was 21.9 and 5.5%, respectively. High PG (≥38 g/L) had sensitivity and specificity of 76.2 and 71.5% respectively for hypergammaglobulinemia. PG ≥38 g/L had a negative predictive value (NPV) of 93.1% for monoclonal, and 96.9% for polyclonal gammopathy. A PG threshold of ≤18 g/L had of sensitivity of 0.4%, specificity of 100%, PPV of 100% and NPV of 80.1% to detect hypogammaglobulinemia (IgG <7 g/L). Conclusions High and low PG values were not sensitive in detecting hyper- or hypogammaglobulinemia, although negative predictive values were high for both. Performance of PG should be further evaluated prospectively in specific populations at risk of for abnormal IgG levels.

Objectives To evaluate the impact of implementing a modified Pediatric Emergency Care Applied Research Network (PECARN) rule including the S100B protein assay for managing mild traumatic brain injury (mTBI) in children. Methods A before-and-after study was conducted in a paediatric emergency department of a French University Hospital from 2013 to 2015. We retrospectively included all consecutive children aged 4 months to 15 years who presented mTBI and were at intermediate risk for clinically important traumatic brain injury (ciTBI). We compared the proportions of CT scans performed and of in-hospital observations before (2013–2014) and after (2014–2015) implementation of a modified PECARN rule including the S100B protein assay. Results We included 1,062 children with mTBI (median age 4.5 years, sex ratio [F/M] 0.73) who were at intermediate risk for ciTBI: 494 (46.5%) during 2013–2014 and 568 (53.5%) during 2014–2015. During 2014–2015, S100B protein was measured in 451 (79.4%) children within 6 h after mTBI. The proportion of CT scans and in-hospital observations significantly decreased between the two periods, from 14.4 to 9.5% (p=0.02) and 73.9–40.5% (p<0.01), respectively. The number of CT scans performed to identify a single ciTBI was reduced by two-thirds, from 18 to 6 CT scans, between 2013–2014 and 2014–2015. All children with ciTBI were identified by the rules. Conclusions The implementation of a modified PECARN rule including the S100B protein assay significantly decreased the proportion of CT scans and in-hospital observations for children with mTBI who were at intermediate risk for ciTBI.

Objectives Autoverification systems have greatly improved laboratory efficiency. However, the long-developed rule-based autoverfication models have limitations. The machine learning (ML) algorithm possesses unique advantages in the evaluation of large datasets. We investigated the utility of ML algorithms for developing an artificial intelligence (AI) autoverification system to support laboratory testing. The accuracy and efficiency of the algorithm model were also validated. Methods Testing data, including 52 testing items with demographic information, were extracted from the laboratory information system and Roche Cobas ® IT 3000 from June 1, 2018 to August 30, 2019. Two rounds of modeling were conducted to train different ML algorithms and test their abilities to distinguish invalid reports. Algorithms with the top three best performances were selected to form the finalized ensemble model. Double-blind testing between experienced laboratory personnel and the AI autoverification system was conducted, and the passing rate and false-negative rate (FNR) were documented. The working efficiency and workload reduction were also analyzed. Results The final AI system showed a 89.60% passing rate and 0.95 per mille FNR, in double-blind testing. The AI system lowered the number of invalid reports by approximately 80% compared to those evaluated by a rule-based engine, and therefore enhanced the working efficiency and reduced the workload in the biochemistry laboratory. Conclusions We confirmed the feasibility of the ML algorithm for autoverification with high accuracy and efficiency.

Objectives The interest around circulating extracellular vesicles and their cargo in diagnostics has greatly increased; however, several pre-analytical variables affect their determination. In this study, we investigated the effects of sample matrix, processing, and plasma storage delay and temperature on extracellular vesicles and their miRNA content. Methods Blood was collected from 10 male volunteers in dipotassium ethylendiaminotetraacetate-coated tubes (K 2 EDTA), either with plasma-preparation tube (PPT) or without (K2E) gel separator. A stepwise centrifugation was applied to K2E aliquots to obtain platelet-poor plasma (PPP). K2E, PPP and PPT plasma, stored under different conditions, were assayed for extracellular vesicles concentration and size distribution, through dynamic laser light scattering, and microRNAs content, by qPCR. Results PPP samples were characterized by the lowest extracellular vesicles count and miRNA detectability. Although having no effects on extracellular vesicles total concentration, storage conditions influenced microRNAs detectability, mainly in PPP and PPT samples. Extracellular vesicles-associated miRNAs levels in K2E were, in general, higher than in PPP and to a very limited extent to PPT. Storage temperature and delay did not affect their profile in K2E samples. Conclusions Extracellular vesicles count and extracellular vesicles miRNA profile changed under the analyzed pre-analytical variables, showing the greatest stability in K2E samples. Since pre-analytical variables differently affected extracellular vesicles and their miRNA content, they should be considered in each experimental setting and clinical routine.

Objectives Pneumatic tube transportation of samples is an effective way of reducing turn-around-time, but evidence of the effect of pneumatic tube transportation on urine samples is lacking. We thus wished to investigate the effect of pneumatic tube transportation on various components in urine, in order to determine if pneumatic tube transportation of these samples is feasible. Methods One-hundred fresh urine samples were collected in outpatient clinics and partitioned with one partition being carried by courier to the laboratory, while the other was sent by pneumatic tube system (Tempus600). Both partitions were then analysed for soluble components and particles, and the resulting mean difference and limits of agreement were calculated. Results Albumin, urea nitrogen, creatinine, protein and squamous epithelial cells were unaffected by transportation in the Tempus600 system, while bacteria, renal tubular epithelial cells, white blood cells and red blood cells were affected and potassium and sodium may have been affected. Conclusions Though pneumatic tube transportation did affect some of the investigated components, in most cases the changes induced were clinically acceptable, and hence samples could be safely transported by the Tempus600 pneumatic tube system. For bacteria, white blood cells and red blood cells local quality demands will determine if pneumatic tube transportation is appropriate.

Objectives Due to its high specificity, liquid chromatography-tandem mass spectrometry (LC-MS/MS) is considered the gold standard in diagnostic areas such as therapeutic monitoring of immunosuppressive drugs (ISDs). However, many laboratories still rely on immunoassays for ISD quantification in a tradeoff between analytical performance and the advantages of fully automated analyzers – shorter turnaround times, greater ease of use, and 24/7 availability. Methods The LC-MS/MS-based Thermo Scientific™ Cascadion™ SM Immunosuppressant Panel was evaluated for >6 months in the routine laboratory of a university hospital. We assessed the analytical performance of the panel and compared it to conventional LC-MS/MS as well as to immunoassays (cyclosporine A, sirolimus, tacrolimus (Siemens) and everolimus (Thermo Fisher)). In addition, both ISD panel and Cascadion analyzer were scrutinized with regards to, e.g., turnaround time, usability, and robustness. Results All ISDs showed high linearity and precision (CV≤6%) and a good correlation with conventional LC-MS/MS. The mean deviation to the immunoassays was 17–19% and negative for all ISDs except everolimus with a positive 19% bias. No weak points were revealed when challenging assay and system with, e.g., high haematocrit, sedimented whole blood or priority samples. The Cascadion integrated well into our 24/7 routine and could easily be operated simultaneously with several other analyzers by technical staff without LC-MS experience. Conclusions The ISD panel showed excellent analytical performance and demonstrated that a fully automated LC-MS-based analysis starting from primary samples is feasible, suggesting that LC-MS could become an integral part of 24/7 diagnostics in the near future.

Objectives Novel treatment options for some carbapenem-resistant Gram-negative pathogens have been identified by the World Health Organization as being of the highest priority. Ceftolozane–tazobactam is a novel cephalosporin–beta-lactamase inhibitor combination antibiotic with potent bactericidal activity against the most difficult-to-treat multi-drug resistant and extensively drug resistant Gram-negative pathogens. This study aimed to develop and validate a liquid chromatography – tandem mass spectrometry method for the simultaneous quantification of ceftolozane and tazobactam in plasma (total and unbound), renal replacement therapy effluent (RRTE), cerebrospinal fluid (CSF) and urine. Methods Analytes were separated using mixed-mode chromatography with an intrinsically base-deactivated C18 column and a gradient mobile phase consisting of 0.1% formic acid, 10 mM ammonium formate and acetonitrile. The analytes and internal standards were detected using rapid ionisation switching between positive and negative modes with simultaneous selected reaction monitoring. Results A quadratic calibration was obtained for plasma (total and unbound), RRTE and CSF over the concentration range of 1–200 mg/L for ceftolozane and 0.5–100 mg/L for tazobactam, and for urine the concentration range of 10–2,000 mg/L for ceftolozane and 5–1,000 mg/L for tazobactam. For both ceftolozane and tazobactam, validation testing for matrix effects, precision and accuracy, specificity and stability were all within the acceptance criteria of ±15%. Conclusions This methodology was successfully applied to one pilot pharmacokinetic study in infected critically ill patients, including patients receiving renal replacement therapy, and one case study of a patient with ventriculitis, where all patients received ceftolozane–tazobactam.

Objectives Therapeutic drug monitoring (TDM) plays a crucial role in personalized medicine. It helps clinicians to tailor drug dosage for optimized therapy through understanding the underlying complex pharmacokinetics and pharmacodynamics. Conventional, non-continuous TDM fails to provide real-time information, which is particularly important for the initial phase of immunosuppressant therapy, e.g., with cyclosporine (CsA) and mycophenolic acid (MPA). Methods We analyzed the time course over 8 h of total and free of immunosuppressive drug (CsA and MPA) concentrations measured by liquid chromatography-tandem mass spectrometry (LC-MS/MS) in 16 kidney transplant patients. Besides repeated blood sampling, intravenous microdialysis was used for continuous sampling. Free drug concentrations were determined from ultracentrifuged EDTA-plasma (UC) and compared with the drug concentrations in the respective microdialysate (µD). µDs were additionally analyzed for free CsA using a novel immunosensor chip integrated into a fluorescence detection platform. The potential of microdialysis coupled with an optical immunosensor for the TDM of immunosuppressants was assessed. Results Using LC-MS/MS, the free concentrations of CsA ( f CsA) and MPA ( f MPA) were detectable and the time courses of total and free CsA comparable. f CsA and f MPA and area-under-the-curves (AUCs) in µDs correlated well with those determined in UCs (r≥0.79 and r≥0.88, respectively). Moreover, f CsA in µDs measured with the immunosensor correlated clearly with those determined by LC-MS/MS (r=0.82). Conclusions The new microdialysis-supported immunosensor allows real-time analysis of immunosuppressants and tailor-made dosing according to the AUC concept. It readily lends itself to future applications as minimally invasive and continuous near-patient TDM.

Objectives Venous blood gas (VBG) analysis is becoming a popular alternative to arterial blood gas (ABG) analysis due to reduced risk of complications at phlebotomy and ease of draw. In lack of published data, this study aimed to establish reference intervals (RI) for correct interpretation of VBG results. Methods One hundred and 51 adult volunteers (101 females, 50 males, 18–70 years) were enrolled after completion of a health questionnaire. Venous blood was drawn into safe PICO syringes and analysed on ABL827 blood gas analyser (Radiometer Pacific Pty. Ltd.). A non-parametric approach was used to directly establish the VBG RI which was compared to a calculated VBG RI based on a meta-analysis of differences between ABG and VBG Results After exclusions, 134 results were used to derive VBG RI: pH 7.30–7.43, partial pressure of carbon dioxide (pCO 2 ) 38–58 mmHg, partial pressure of oxygen (pO 2 ) 19–65 mmHg, bicarbonate (HCO 3 −) 22–30 mmol/L, sodium 135–143 mmol/L, potassium 3.6–4.5 mmol/L, chloride 101–110 mmol/L, ionised calcium 1.14–1.29 mmol/L, lactate 0.4–2.2 mmol/L, base excess (BE) −1.9–4.5 mmol/L, saturated oxygen (sO 2 ) 23–93%, carboxyhaemoglobin 0.4–1.4% and methaemoglobin 0.3–0.9%. The meta-analysis revealed differences between ABG and VBG for pH, HCO 3 −, pCO 2 and pO 2 of 0.032, −1.0 mmol/L, −4.2 and 39.9 mmHg, respectively. Using this data along with established ABG RI, calculated VBG RI of pH 7.32–7.42, HCO 3 − 23 – 27 mmol/L, pCO 2 36–49 mmHg (female), pCO 2 39–52 mmHg (male) and pO 2 43–68 mmHg were formulated and compared to the VBG RI of this study. Conclusions An adult reference interval has been established to assist interpretation of VBG results.

Objectives Dilated cardiomyopathy (DCM) represents the most frequent form of cardiomyopathy, leading to heart failure, cardiac arrhythmias and death. Accumulating evidence convincingly demonstrates the crucial role of genetic defects in the pathogenesis of DCM, and over 100 culprit genes have been implicated with DCM. However, DCM is of substantial genetic heterogeneity, and the genetic determinants underpinning DCM remain largely elusive. Methods Whole-exome sequencing and bioinformatical analyses were implemented in a consanguineous Chinese family with DCM. A total of 380 clinically annotated control individuals and 166 more DCM index cases then underwent Sanger sequencing analysis for the identified genetic variation. The functional characteristics of the variant were delineated by utilizing a dual-luciferase assay system. Results A heterozygous variation in the MEF2A gene (encoding myocyte enhancer factor 2A, a transcription factor pivotal for embryonic cardiogenesis and postnatal cardiac adaptation), NM_001365204.1: c.718G>T; p. (Gly240*), was identified, and verified by Sanger sequencing to segregate with autosome-dominant DCM in the family with complete penetrance. The nonsense variation was neither detected in 760 control chromosomes nor found in 166 more DCM probands. Functional analyses revealed that the variant lost transactivation on the validated target genes MYH6 and FHL2 , both causally linked to DCM. Furthermore, the variation nullified the synergistic activation between MEF2A and GATA4, another key transcription factor involved in DCM. Conclusions The findings firstly indicate that MEF2A loss-of-function variation predisposes to DCM in humans, providing novel insight into the molecular mechanisms of DCM and suggesting potential implications for genetic testing and prognostic evaluation of DCM patients.

Objectives Hemoglobin A 1c (HbA 1c ) is a valuable parameter in the monitoring of diabetic patients and increasingly in diagnosis of diabetes. Manufacturers continuously optimize instruments, currently the main focus is to achieve faster turnaround times. It is important that performance specifications remain of high enough standard, which is evaluated in this study for the new ARKRAY HA-8190V instrument. Methods The Clinical and Laboratory Standards Institute (CLSI) protocols EP-5, EP-9 and EP-10 were applied to investigate imprecision, bias and linearity. In addition potential interferences, performance in External Quality Assessment (EQA) and performance against the HA-8180V instrument in 220 clinical samples was evaluated. Results The HA-8190V demonstrates a CV of ≤0.8% in IFCC SI units (≤0.6% National Glycohemoglobin Standardization Program [NGSP]) at 34 and 102 mmol/mol levels (5.3 and 11.5% NGSP) and a bias of −0.1 mmol/mol (−0.01% NGSP) at a concentration of 50 mmol/mol (6.7% NGSP), but with a significant slope as compared to target values. This results in a bias of −1.0 and 0.9 mmol/mol (−2.0 and 0.9% NGSP) at the 30 and 70 mmol/mol (4.9 and 8.6% NGSP) concentration level. Simulation of participation in the IFCC certification programme results in a Silver score (bias −0.1 mmol/mol, CV 1.1%). Interference in the presence of the most important Hb variants (AS, AC, AE, AD) and elevated HbA 2 and HbF concentrations is less than 3 mmol/mol (0.3% NGSP) at a concentration of 50 mmol/mol (6.7% NGSP). Conclusions Analytical performance of the HA-8190V is very good, especially with respect to precision and HbA 1c quantification in the presence of the most common Hb variants.

Objectives Accurate SARS-CoV-2 serological assays are urgently needed to help diagnose infection, determine past exposure of populations and assess the response to future vaccines. The study aims at assessing the performance of the multiplex D-tek COVIDOT 5 IgG assay for the detection of SARS-CoV-2 IgG antibodies (N, S1+S2, S1, S2 and RBD). Methods Sensitivity and dynamic trend to seropositivity were evaluated in 218 samples obtained from 46 rRT-PCR confirmed COVID-19 patients. Non-SARS-CoV-2 sera (n=118) collected before the COVID-19 pandemic with a potential cross-reaction to the SARS-CoV-2 immunoassay were included in the specificity analysis. Results A gradual dynamic trend since symptom onset was observed for all IgG antibodies. Sensitivities before day 14 were suboptimal. At ≥21 days, sensitivities reached 100% (93.4–100%) for N, S1+S2, S2 and RBD-directed IgG and 96.3% (87.3–99.6%) for S1-directed IgG. In 42 out of 46 patients (91.3%), all five antibodies were detected at ≥14 days. The four remaining patients had between 2 and 4 positive antibodies at their respective maximal follow-up period. The specificity was 100 % for S1+S2, S2 and RBD, 98.3% for N and 92.4% (86.0–96.5%) for S1-directed IgG. The combined use of antigens increases the early sensitivity whilst enforcing high specificity. Conclusions Sensitivities at ≥21 days and specificities were excellent, especially for N, S1+S2, S2 and RBD-directed IgG. Caution is however required when interpreting single S1-directed reactivities. Using a multiplex assay complies with the orthogonal testing algorithm of the CDC and allows a better and critical interpretation of the serological status of a patient.

Objectives During the current pandemic, antibody testing based on venous serum helps to determine whether the tested person has been previously infected with SARS-CoV-2. Alternatively, capillary blood can be taken via a finger prick (dried blood spots, DBS). In this study, paired DBS and venipuncture samples were tested using two serological assays to evaluate the usability of DBS for the detection of anti-SARS-CoV-2 antibodies. Methods Paired samples of DBS and venous serum were collected from 389 volunteers, of whom 75 had a recent PCR-confirmed SARS-CoV-2 infection, and tested for anti-SARS-CoV-2 IgG antibodies against both viral S1 and nucleocapsid protein (NCP) antigens using two ELISAs. Degree of agreement and correlation coefficients between ELISA results based on the two sampling methods were calculated. Results Results of DBS showed almost perfect agreement and high correlations with results from corresponding serum samples in both the S1-based ELISA and the NCP-based ELISA. Conclusions ELISA results derived from DBS showed very high agreement to those obtained with serum, supposing adequate usability and robustness of DBS as sample material for detection of anti-SARS-CoV-2 antibodies. In the near future, large-scale epidemiological screening for antibodies against SARS-CoV-2 will be carried out. Since DBS reduce the strain on healthcare institutions regarding sample collection, they have a potential to facilitate efficient community- and population-based screening in the current SARS-CoV-2 pandemic.

Objectives The qualitative results of SARS-CoV-2 specific real-time reverse transcription (RT) PCR are used for initial diagnosis and follow-up of Covid-19 patients and asymptomatic virus carriers. However, clinical decision-making and health management policies often are based additionally on cycle threshold (C t ) values (i.e., quantitative results) to guide patient care, segregation and discharge management of individuals testing positive. Therefore, an analysis of inter-protocol variability is needed to assess the comparability of the quantitative results. Methods C t values reported in a SARS-CoV-2 virus genome detection external quality assessment challenge were analyzed. Three positive and two negative samples were distributed to participating test laboratories. Qualitative results (positive/negative) and quantitative results (C t values) were assessed. Results A total of 66 laboratories participated, contributing results from 101 distinct test systems and reporting C t values for a total of 92 different protocols. In all three positive samples, the means of the C t values for the E -, N -, S -, RdRp -, and ORF1ab -genes varied by less than two cycles. However, 7.7% of reported results deviated by more than ±4.0 (maximum 18.0) cycles from the respective individual means. These larger deviations appear to be systematic errors. Conclusions In an attempt to use PCR diagnostics beyond the identification of infected individuals, laboratories are frequently requested to report C t values along with a qualitative result. This study highlights the limitations of interpreting C t values from the various SARS-CoV genome detection protocols and suggests that standardization is necessary in the reporting of C t values with respect to the target gene.

Objectives Midregional pro-adrenomedullin (MR-proADM) is a vasoactive peptide with key roles in reducing vascular hyperpermeability and thereby improving endothelial stability during infection. While MR-proADM is useful for risk stratification in patients with sepsis, clinical data about prediction accuracy in patients with severe acute respiratory syndrome coronavirus 2 disease (COVID-19) is currently missing. Methods We included consecutively adult patients hospitalized for confirmed COVID-19 at a tertiary care center in Switzerland between February and April 2020. We investigated the association of MR-proADM levels with in-hospital mortality in logistic regression and discrimination analyses. Results Of 89 included COVID-19 patients, 19% (n=17) died while in the hospital. Median admission MR-proADM levels (nmol/L) were increased almost 1.5-fold increased in non-survivors compared to survivors (1.3 [interquartile range IQR 1.1–2.3]) vs. 0.8 [IQR 0.7–1.1]) and showed good discrimination (area under the curve 0.78). An increase of 1 nmol/L of admission MR-proADM was independently associated with a more than fivefold increase in in-hospital mortality (adjusted odds ratio of 5.5, 95% confidence interval 1.4–21.4, p=0.015). An admission MR-proADM threshold of 0.93 nmol/L showed the best prognostic accuracy for in-hospital mortality with a sensitivity of 93%, a specificity of 60% and a negative predictive value of 97%. Kinetics of follow-up MR-proADM provided further prognostic information for in-hospital treatment. Conclusions Increased levels of MR-proADM on admission and during hospital stay were independently associated with in-hospital mortality and may allow a better risk stratification, and particularly rule-out of fatal outcome, in COVID-19 patients.