To rule-in, or not to falsely rule-out, that is the question: evaluation of hs-cTnT EQA performance in light of the ESC-2020 guideline.

OBJECTIVES: To accurately evaluate non-ST-elevated acute cardiac syndrome (NSTE-ACS), the quality of high-sensitive cardiac troponin (hs-cTn) assays is of vital importance. The 2020 revision of the NSTE-ACS guideline includes clinical decision-limits (CDL's) to both rule-in and rule-out NSTE-ACS for most commercially available platforms, providing both 0/1 h and 0/2 h delta limits. Our study evaluated whether laboratories are able to meet the analytical performance specifications for imprecision (APS) for hs-cTnT. METHODS: Results from external quality assurance (EQA) in commutable samples were used to evaluate the current and historic performance of analyzers. The performance of analyzers that either passed or failed to comply with 0/1 h-APS were used on a real-world dataset of first hs-cTnT-values to simulate 10.000 samples of t=0, t=1 and t=2 h values with multiple delta's for all relevant CDL's. We compared the simulated values to the input values to obtain the percentage of aberrant results simulated. RESULTS: The majority of analyzers complies with APS for rule-in in 2022 (0/1 h: 90.4 % and 0/2 h: 100 %), compliance for the 0/1 h rule-out is still far from optimal (0/1 h: 30.7 %, 0/2 h: 75.4 %), with improving compliance over the past years (rule-in p=<0.0001, rule-out p=0.011, χ2). Whilst 0/1 h-APS-passing analyzers have a minute risk to falsely rule-out patients whom should be ruled-in (0.0001 %), failing performance increases this risk to 2.1 % upon using 0/1 h CDL's. Here, adopting 0/2 h CDL's is favorable (0.01 %). CONCLUSIONS: Laboratories that fail to meet hs-cTnT 0/1 h-APS should improve their performance to the required and achievable level. Until performance is reached clinics should adopt the 0/2 h CDL's.

A Significant Increase in the Incidence of Neonatal Hyperbilirubinemia and Phototherapy Treatment Due to a Routine Change in Laboratory Equipment.

CONTEXT.­: Total serum bilirubin (TSB) analysis is pivotal for diagnosing neonatal hyperbilirubinemia. Because of a routine change in laboratory equipment, our TSB assay changed from a diazo to a vanadate oxidase method. Upon implementation, TSB results were substantially higher in newborns than expected based on the validation. OBJECTIVE.­: To investigate the application of TSB and intermethod differences in neonates and their impact on phototherapy treatment. DESIGN.­: The diazo and vanadate methods were compared directly using neonatal and adult samples. Anonymized external quality control data were analyzed to explore interlaboratory differences among 8 commercial TSB assays. Clinical patient data were extracted from the medical records to investigate the number of newborns receiving phototherapy. RESULTS.­: The mean bias of the vanadate versus the diazo TSB method was +17.4% and +3.7% in neonatal and adult samples, respectively. External quality control data showed that the bias of commercial TSB methods compared with the reference method varied from -3.6% to +20.2%. Within-method variation ranged from 5.2% to 16.0%. After implementation of the vanadate TSB method, the number of neonates treated with phototherapy increased approximately threefold. CONCLUSIONS.­: Currently available TSB assays lack harmonization for the diagnosis of neonatal hyperbilirubinemia. Between-methods differences are substantially higher in neonatal compared with adult samples, highlighting the importance of including neonatal samples during assay validation. Close collaboration between laboratory specialists and clinicians is essential to prevent overtreatment or undertreatment upon the implementation of novel analyzers or assays. Also, harmonization of TSB assays, with an emphasis on neonatal application, is warranted.

Albumin determined by bromocresol green leads to erroneous results in routine evaluation of patients with chronic kidney disease.

OBJECTIVES: Measurement of plasma albumin is pivotal for clinical decision-making in patients with chronic kidney disease (CKD). Routinely used methods as bromocresol green (BCG) and bromocresol purple (BCP) can suffer from aselectivity, but the impact of aselectivity on the accuracy of plasma albumin results of CKD-patients is still unknown. Therefore, we evaluated the performance of BCG-, BCP- and JCTLM-endorsed immunological methods in patients with various stages of CKD. METHODS: We evaluated the performance of commonly used albumin methods in patients with CKD stages G1 through G5, the latter divided in two groups based on whether they received hemodialysis treatment. In total, 163 patient plasma samples were measured at 14 laboratories, on six different BCG and BCP-platforms, and four different immunological platforms. The results were compared with an ERM-DA-470k-corrected nephelometric assay. The implications on outcome is evaluated by the proportion of patient results <38 g/L for the diagnosis of protein energy wasting. RESULTS: Albumin results determined with BCP- and immunological methods showed the best agreement with the target value (92.7 and 86.2 %, respectively vs. 66.7 % for BCG, namely due to overestimation). The relative agreement of each method with the target value was platform-dependent, with larger variability in agreement between platforms noted for BCG and immunological methods (3.2-4.6 and 2.6-5.3 %) as opposed to BCP (0.7-1.5 %). The stage of CKD had similar effects on the variability in agreement for the three method-groups (0.6-1.8 % vs. 0.7-1.5 % vs. 0.4-1.6 %). The differences between methods cause discrepancies in clinical decision-making, as structurally fewer patients were diagnosed with protein energy wasting upon using BCG-based albumin results. CONCLUSIONS: Our study shows that BCP is fit for the intended use to measure plasma albumin levels in CKD patients from all stages, including patients on hemodialysis. In contrast, most BCG-based platforms falsely overestimate the plasma albumin concentration.

Serum albumin measurement in nephrology: room for improvement.

Serum albumin is a widely used biomarker in clinical nephrology. Serum albumin cut-off values are used to define disease, to predict outcome and to guide patient care. The available commercial assays to measure serum albumin rely on different analytical principles, all with their own (analytical) specifications. This article provides an overview of the different clinical applications of serum albumin measurements in nephrology, the (dis)advantages of the available assays and the estimates of the effects of the measurement uncertainty between different assays in clinical decision making. This article concludes that harmonization of serum albumin assay results is needed.

Dexamethasone and tocilizumab treatment considerably reduces the value of C-reactive protein and procalcitonin to detect secondary bacterial infections in COVID-19 patients.

BACKGROUND: Procalcitonin (PCT) and C-reactive protein (CRP) were previously shown to have value for the detection of secondary infections in critically ill COVID-19 patients. However, since the introduction of immunomodulatory therapy, the value of these biomarkers is unclear. We investigated PCT and CRP kinetics in critically ill COVID-19 patients treated with dexamethasone with or without tocilizumab, and assessed the value of these biomarkers to detect secondary bacterial infections. METHODS: In this prospective study, 190 critically ill COVID-19 patients were divided into three treatment groups: no dexamethasone, no tocilizumab (D-T-), dexamethasone, no tocilizumab (D+T-), and dexamethasone and tocilizumab (D+T+). Serial data of PCT and CRP were aligned on the last day of dexamethasone treatment, and kinetics of these biomarkers were analyzed between 6 days prior to cessation of dexamethasone and 10 days afterwards. Furthermore, the D+T- and D+T+ groups were subdivided into secondary infection and no-secondary infection groups to analyze differences in PCT and CRP kinetics and calculate detection accuracy of these biomarkers for the occurrence of a secondary infection. RESULTS: Following cessation of dexamethasone, there was a rebound in PCT and CRP levels, most pronounced in the D+T- group. Upon occurrence of a secondary infection, no significant increase in PCT and CRP levels was observed in the D+T- group (p = 0.052 and p = 0.08, respectively). Although PCT levels increased significantly in patients of the D+T+ group who developed a secondary infection (p = 0.0003), this rise was only apparent from day 2 post-infection onwards. CRP levels remained suppressed in the D+T+ group. Receiver operating curve analysis of PCT and CRP levels yielded area under the curves of 0.52 and 0.55, respectively, which are both markedly lower than those found in the group of COVID-19 patients not treated with immunomodulatory drugs (0.80 and 0.76, respectively, with p values for differences between groups of 0.001 and 0.02, respectively). CONCLUSIONS: Cessation of dexamethasone in critically ill COVID-19 patients results in a rebound increase in PCT and CRP levels unrelated to the occurrence of secondary bacterial infections. Furthermore, immunomodulatory treatment with dexamethasone and tocilizumab considerably reduces the value of PCT and CRP for detection of secondary infections in COVID-19 patients.

Serum Magnesium Is Inversely Associated With Heart Failure, Atrial Fibrillation, and Microvascular Complications in Type 2 Diabetes.

OBJECTIVE: We investigated whether serum magnesium (Mg2+) was prospectively associated with macro- or microvascular complications and mediated by glycemic control (hemoglobin A1c [HbA1c]), in type 2 diabetes (T2D). RESEARCH DESIGN AND METHODS: We analyzed in 4,348 participants the association of serum Mg2+ with macrovascular disease and mortality (acute myocardial infarction [AMI], coronary heart disease [CHD], heart failure [HF], cerebrovascular accident [CVA], and peripheral arterial disease [PAD]), atrial fibrillation (AF), and microvascular complications (chronic kidney disease [CKD], diabetic retinopathy, and diabetic foot) using Cox regression, adjusted for confounders. Mediation analysis was performed to assess whether HbA1c mediated these associations. RESULTS: The average baseline serum Mg2+ concentration was 0.80 ± 0.08 mmol/L. During 6.1 years of follow-up, serum Mg2+ was inversely associated with major macrovascular, 0.87 (95% CI 0.76; 1.00); HF, 0.76 (95% CI 0.62; 0.93); and AF, 0.59 (95% CI 0.49; 0.72). Serum Mg2+ was not associated with AMI, CHD, CVA, and PAD. During 5.1 years of follow-up, serum Mg2+ was inversely associated with overall microvascular events, 0.85 (95% CI 0.78; 0.91); 0.89 (95% CI 0.82; 0.96) for CKD, 0.77 (95% CI 0.61; 0.98) for diabetic retinopathy, and 0.85 (95% CI 0.78; 0.92) for diabetic foot. HbA1c mediated the associations of serum Mg2+ with HF, overall microvascular events, diabetic retinopathy, and diabetic foot. CONCLUSIONS: Serum Mg2+ concentration is inversely associated with the risk to develop HF and AF and with the occurrence of CKD, diabetic retinopathy, and foot complications in T2D. Glycemic control partially mediated the association of serum Mg2+ with HF and microvascular complications.

SGLT2 inhibition versus sulfonylurea treatment effects on electrolyte and acid-base balance: secondary analysis of a clinical trial reaching glycemic equipoise: Tubular effects of SGLT2 inhibition in Type 2 diabetes.

Sodium-glucose transporter (SGLT)2 inhibitors increase plasma magnesium and plasma phosphate and may cause ketoacidosis, but the contribution of improved glycemic control to these observations as well as effects on other electrolytes and acid-base parameters remain unknown. Therefore, our objective was to compare the effects of SGLT2 inhibitors dapagliflozin and sulfonylurea gliclazide on plasma electrolytes, urinary electrolyte excretion, and acid-base balance in people with Type 2 diabetes (T2D). We assessed the effects of dapagliflozin and gliclazide treatment on plasma electrolytes and bicarbonate, 24-hour urinary pH and excretions of electrolytes, ammonium, citrate, and sulfate in 44 metformin-treated people with T2D and preserved kidney function. Compared with gliclazide, dapagliflozin increased plasma chloride by 1.4 mmol/l (95% CI 0.4-2.4), plasma magnesium by 0.03 mmol/l (95% CI 0.01-0.06), and plasma sulfate by 0.02 mmol/l (95% CI 0.01-0.04). Compared with baseline, dapagliflozin also significantly increased plasma phosphate, but the same trend was observed with gliclazide. From baseline to week 12, dapagliflozin increased the urinary excretion of citrate by 0.93 ± 1.72 mmol/day, acetoacetate by 48 µmol/day (IQR 17-138), and ß-hydroxybutyrate by 59 µmol/day (IQR 0-336), without disturbing acid-base balance. In conclusion, dapagliflozin increases plasma magnesium, chloride, and sulfate compared with gliclazide, while reaching similar glucose-lowering in people with T2D. Dapagliflozin also increases urinary ketone excretion without changing acid-base balance. Therefore, the increase in urinary citrate excretion by dapagliflozin may reflect an effect on cellular metabolism including the tricarboxylic acid cycle. This potentially contributes to kidney protection.

The bias between different albumin assays may affect clinical decision-making.

Differences between laboratory assays can have important clinical implications. For creatinine assays this became apparent soon after the introduction of the Modification of Diet in Renal Disease formula and resulted in international efforts towards standardization. Albumin in blood is measured by different assays, either chromogenic using Bromocresol green (BCG) or Bromocresol purple (BCP), or by an immunoassay. Since differences between these assays have received limited attention we evaluated bias and imprecision of BCG and BCP assays in comparison to the immunoassay using blood samples from patients with membranous nephropathy and nephrotic syndrome. For the BCG assay, the mean bias was high (6.2 g/l, with a standard deviation of 2.4 g/l) compared to a bias of 0.3 g/l (standard deviation 1.5 g/l) for the BCP assay. Importantly, we questioned clinical relevance by evaluating the accuracy of the decision toward the use of prophylactic anticoagulant therapy. Notably, nephrologists may reach inappropriate treatment decisions using the BGC assay in up to 59% of patients. Thus, our study should stimulate efforts towards standardization of the albumin assays.

Diagnosis of acute myocardial infarction in hemodialysis patients may be feasible by comparing variation of cardiac troponins during acute presentation to baseline variation.

Acute myocardial infarction (AMI) is defined as a dynamic change in cardiac troponin (cTn) with at least one cTn value exceeding the 99 th percentile of a reference population in combination with typical clinical symptoms. In hemodialysis (HD) patients, a broad range of cTn concentrations is found, partially due to patient-specific comorbidities. Therefore, the 99 th percentile cannot be used in HD patients and decision algorithms to diagnose AMI should be based on temporal variations of troponin. In this study, relative and absolute variations of cTn in a large population of asymptomatic hemodialysis patients were established during a period of 15 months. Patients were stratified according to their history of coronary artery disease (CAD). An intra-individual long term variation of 23% for cTroponin I (cTnI) and 12% for cTroponin T (cTnT) was found for patients without a history of CAD. The corresponding reference change values (RCVs) were 69% and 39% respectively. For patients with a history of CAD this variation was 29% for cTnI and 10% for cTnT, with RCVs of 86% and 35% respectively. During follow up, 27 HD patients developed an acute myocardial infarction (AMI). During these events, irrespective of CAD history, cTnI increased>172% and cTnT increased>97% above baseline cTn as measured during clinically stable periods three months separate to the event. Therefore, if a HD patient has symptoms of an acute event and a cTn increase that exceeds the RCVs described here, AMI may be suspected. If the troponin increase exceeds 172% for cTnI or 97% for cTnT, AMI is likely.

Moderate elevations of high-sensitivity cardiac troponin I and B-type natriuretic peptide in chronic hemodialysis patients are associated with mortality.

BACKGROUND: Several biomarkers are associated with mortality in hemodialysis patients. In particular, elevated cardiac troponin T and B-type natriuretic peptide (BNP) are strong predictors of mortality; however, less is known about cardiac troponin I (cTnI). Elevated troponin I is detected in many hemodialysis patients, but the association of moderate elevations with mortality is unclear. METHODS: The relation between mortality and cTnI, using a high-sensitivity cTnI assay, as well as BNP and C-reactive protein (CRP) was evaluated in 206 chronic hemodialysis patients. RESULTS: Median follow-up was 28 months with a total mortality of 35%. Mortality was significantly associated with elevated cTnI, BNP and CRP. Even patients with only moderate elevation of cTnI (0.01-0.10 µg/L) showed 2.5-fold increased mortality. Interestingly, hazard ratios for mortality for single (random) measurements were comparable to those for mean/median measurements. Subsequently, subgroup analysis based on combined markers was performed. Patients with both cTnI <0.01 µg/L and BNP in the first quartile had 100% survival. Patients with either cTnI <0.01 µg/L or BNP in the lowest quartile had significantly lower mortality (12% and 13%, respectively) than patients with BNP levels in the second quartile or higher and cTnI of 0.01-0.05 µg/L and patients with cTnI ≥0.05 µg/L (mortality 46 and 58%, respectively). CONCLUSIONS: A combination of moderate elevation of cTnI and BNP provided additional prognostic value. A single measurement of these biomarkers performed comparably to the mean/median of multiple measurements.

Immune responses against domain I of ß(2)-glycoprotein I are driven by conformational changes: domain I of ß(2)-glycoprotein I harbors a cryptic immunogenic epitope.

OBJECTIVE: The presence of autoantibodies against a cryptic epitope in domain I of ß(2)-glycoprotein I (ß(2)GPI) is strongly associated with thrombotic events in patients with the antiphospholipid syndrome. We hypothesized that a conformational change could be a trigger for the formation of antibodies against domain I of ß(2)GPI. Therefore, we investigated whether immune responses against ß(2)GPI are related to its conformation. METHODS: Conformational changes in ß(2)GPI were studied using various techniques, either upon binding to cardiolipin or after disruption of the internal disulfide bonds. The immunogenicity of ß(2)GPI in different conformations as well as the individual domains of ß(2)GPI were studied in vivo by monitoring the generation of antibodies after intravenous administration of ß(2)GPI to mice. Furthermore, plasma samples from these mice were assessed for lupus anticoagulant activity and thrombin-antithrombin complex levels. RESULTS: We observed that the interaction of ß(2)GPI with cardiolipin induced a conformational change in ß(2)GPI: electron microscopy revealed that ß(2)GPI assembled into polymeric meshworks. We next investigated the immunogenicity of both human and murine ß(2)GPI in mice. Both human and murine ß(2)GPI combined with cardiolipin and misfolded ß(2)GPI triggered antibody formation against the native protein as well as against domain I of ß(2)GPI, while native ß(2)GPI was not immunogenic. In addition, we observed that anti-domain I antibodies developed in mice injected with domain I of ß(2)GPI, and that antibodies did not develop in mice injected with domains II-V. The induced anti-domain I antibodies prolonged the dilute Russell's viper venom plasma clotting time. The plasma of mice with anti-domain I antibodies had increased levels of circulating thrombin-antithrombin complexes. CONCLUSION: The results of our studies indicate that the exposure of cryptic epitopes due to conformational changes in ß(2)GPI can induce autoantibody formation.

Electrolyte-balanced heparin in blood gas syringes can introduce a significant bias in the measurement of positively charged electrolytes.

BACKGROUND: Heparin binds positively charged electrolytes. In blood gas syringes, electrolyte-balanced heparin is used to prevent a negative bias in electrolyte concentrations. The potential pre-analytical errors introduced by blood gas syringes are largely unknown. Here, we evaluate electrolyte concentrations in non-anticoagulated blood compared with concentrations measured in electrolyte-balanced blood gas syringes. METHODS: Venous blood was collected into plain tubes. Ionized calcium, potassium, sodium and hydrogen ions were analyzed directly using a blood gas analyzer and the remaining blood was collected into different blood gas syringes in random order: Preset (Becton Dickinson), Monovette (Sarstedt) and Pico 50-2 (Radiometer). RESULTS: Ionized calcium and sodium concentrations were significantly lower in blood collected in Becton Dickinson and Sarstedt syringes compared to non-heparinized (NH) blood. The mean bias exceeded biological variation-based total allowable error, which in most cases leads to clinically misleading individual results. In contrast, ionized calcium concentrations in blood collected in Pico 50-2 syringes were identical to values obtained from NH blood. Sodium showed a minor, yet statistically significant, bias. CONCLUSIONS: Despite the fact that blood gas syringes now contain electrolyte-balanced heparin, one should be aware of the fact that these syringes can introduce pre-analytical bias in electrolyte concentrations. The extent of the bias differs between syringes.

A critical contribution of both CD28 and ICOS in the adjuvant activity of Neisseria meningitidis H44/76 LPS and lpxL1 LPS.

The development of novel vaccines against Neisseria meningitidis recently gained momentum by the generation of penta-acylated lpxL1 LPS which has similar adjuvant activity, but reduced endotoxic activity as compared to hexa-acylated wild type (H44/76) LPS. We investigated the costimulation requirements for the adjuvant activity of both forms of LPS by immunizing CD28-, ICOS- and B7.1/2/ICOS-deficient mice. Both ICOS and CD28 appeared essential for optimal adjuvant activity of H44/76 LPS or lpxL1 LPS. Interestingly, ICOS-mediated costimulation predominates in the adjuvant activity of lpxL1 LPS, while both ICOS and CD28 are required for H44/76 LPS adjuvant activity.

CD28 and ICOS: similar or separate costimulators of T cells?

Numerous studies have revealed that the B7.1/B7.2-CD28 and B7RP-1-ICOS (Inducible COStimulator) pathways provide crucial costimulatory signals to T cells. We have compared the contribution of these pathways during primary and effector responses, in vitro and in vivo, molecularly as well as functionally. This comparison between CD28 an ICOS after initiation of T cell activation demonstrates that both CD28 and ICOS function similarly during expansion, survival and differentiation of T cells and that both CD28 and ICOS are necessary for proper IgG responses. The major differences between CD28 and ICOS are differences in expression of both receptors and ligands, and the fact that CD28 induces IL-2 production, whereas ICOS does not. In addition, ICOS is more potent in the induction of IL-10 production, a cytokine important for suppressive function of T regulatory cells. All data available at present indicate that both molecules are very suitable candidates for immunotherapy, each in their own unique way.