Tumor necrosis factor α promotes clonal dominance of KIT D816V+ cells in mastocytosis: role of survivin and impact on prognosis.

ABSTRACT: Systemic mastocytosis (SM) is defined by the expansion and accumulation of neoplastic mast cells (MCs) in the bone marrow (BM) and extracutaneous organs. Most patients harbor a somatic KIT D816V mutation, which leads to growth factor-independent KIT activation and accumulation of MC. Tumor necrosis factor α (TNF) is a proapoptotic and inflammatory cytokine that has been implicated in the clonal selection of neoplastic cells. We found that KIT D816V increases the expression and secretion of TNF. TNF expression in neoplastic MCs is reduced by KIT-targeting drugs. Similarly, knockdown of KIT or targeting the downstream signaling cascade of MAPK and NF-κB signaling reduced TNF expression levels. TNF reduces colony formation in human BM cells, whereas KIT D816V+ cells are less susceptible to the cytokine, potentially contributing to clonal selection. In line, knockout of TNF in neoplastic MC prolonged survival and reduced myelosuppression in a murine xenotransplantation model. Mechanistic studies revealed that the relative resistance of KIT D816V+ cells to TNF is mediated by the apoptosis-regulator BIRC5 (survivin). Expression of BIRC5 in neoplastic MC was confirmed by immunohistochemistry of samples from patients with SM. TNF serum levels are significantly elevated in patients with SM and high TNF levels were identified as a biomarker associated with inferior survival. We here characterized TNF as a KIT D816V-dependent cytokine that promotes clonal dominance. We propose TNF and apoptosis-associated proteins as potential therapeutic targets in SM.

Hereditary α tryptasemia is a valid genetic biomarker for severe mediator-related symptoms in mastocytosis.

Mastocytosis is a hematopoietic neoplasm characterized by expansion of KIT D816V-mutated clonal mast cells in various organs and severe or even life-threatening anaphylactic reactions. Recently, hereditary α-tryptasemia (HαT) has been described as a common genetic trait with increased copy numbers of the α-tryptase encoding gene, TPSAB1, and associated with an increased basal serum tryptase level and a risk of mast cell activation. The purpose of our study was to elucidate the clinical relevance of HαT in patients with mastocytosis. TPSAB1 germline copy number variants were assessed by digital polymerase chain reaction in 180 mastocytosis patients, 180 sex-matched control subjects, 720 patients with other myeloid neoplasms, and 61 additional mastocytosis patients of an independent validation cohort. α-Tryptase encoding TPSAB1 copy number gains, compatible with HαT, were identified in 17.2% of mastocytosis patients and 4.4% of the control population (P < .001). Patients with HαT exhibited higher tryptase levels than patients without HαT (median tryptase in HαT+ cases: 49.6 ng/mL vs HαT- cases: 34.5 ng/mL, P = .004) independent of the mast cell burden. Hymenoptera venom hypersensitivity reactions and severe cardiovascular mediator-related symptoms/anaphylaxis were by far more frequently observed in mastocytosis patients with HαT than in those without HαT. Results were confirmed in an independent validation cohort. The high prevalence of HαT in mastocytosis hints at a potential pathogenic role of germline α-tryptase encoding TPSAB1 copy number gains in disease evolution. Together, our data suggest that HαT is a novel emerging robust biomarker in mastocytosis that is useful for determining the individual patient´s risk of developing severe anaphylaxis.

Serum sPD-L1 levels are elevated in patients with viral diseases, bacterial sepsis or in patients with impaired renal function compared to healthy blood donors.

OBJECTIVES: Immune checkpoints play an important role in maintaining the balance of the immune system and in the development of autoimmune diseases. A central checkpoint molecule is the programmed cell death protein 1 (PD-1, CD279) which is typically located on the surface of T cells. Its primary ligand PD-L1 is expressed on antigen presenting cells and on cancer cells. Several variants of PD-L1 exist, among these soluble molecules (sPD-L1) present in serum at low concentrations. sPD-L1 was found elevated in cancer and several other diseases. sPD-L1 in infectious diseases has received relatively little attention so far and is therefore subject of this study. METHODS: sPD-L1 serum levels were determined in 170 patients with viral infections (influenza, varicella, measles, Dengue fever, SARS-CoV2) or bacterial sepsis by ELISA and compared to the levels obtained in 11 healthy controls. RESULTS: Patients with viral infections and bacterial sepsis generally show significantly higher sPD-L1 serum levels compared to healthy donors, except for varicella samples where results do not reach significance. sPD-L1 is increased in patients with impaired renal function compared to those with normal renal function, and sPD-L1 correlates significantly with serum creatinine. Among sepsis patients with normal renal function, sPD-L1 serum levels are significantly higher in Gram-negative sepsis compared to Gram-positive sepsis. In addition, in sepsis patients with impaired renal function, sPD-L1 correlates positively with ferritin and negatively with transferrin. CONCLUSIONS: sPD-L1 serum levels are significantly elevated in patients with sepsis, influenza, mesasles, Dengue fever or SARS-CoV2. Highest levels are detectable in patients with measles and Dengue fever. Also impaired renal function causes an increase in levels of sPD-L1. As a consequence, renal function has to be taken into account in the interpretation of sPD-L1 levels in patients.

Molecular quantification of tissue disease burden is a new biomarker and independent predictor of survival in mastocytosis.

A high allele burden of the KIT D816V mutation in peripheral blood or bone marrow aspirates indicates multi-lineage hematopoietic involvement and has been associated with an aggressive clinical course of systemic mastocytosis. Since mast cells are substantially underrepresented in these liquid specimens, their mutation burden likely underestimates the tumor burden of the disease. We used a novel previously validated digital polymerase chain reaction (PCR) method for KIT D816V analysis to systematically analyze the mutation burden in formalin-fixed, paraffin-embedded bone marrow tissue sections of 116 mastocytosis patients (91 with indolent and 25 with advanced systemic mastocytosis), and to evaluate for the first time the clinical value of the tissue mutation burden as a novel biomarker. The KIT D816V mutation burden in the tissue was significantly higher and correlated better with bone marrow mast cell infiltration (r=0.68 vs 0.48) and serum tryptase levels (r=0.68 vs 0.58) compared to that in liquid specimens. Furthermore, the KIT D816V tissue mutation burden was: (i) significantly higher in advanced than in indolent systemic mastocytosis (P=0.001); (ii) predicted survival of patients in multivariate analyses independently; and (iii) was significantly reduced after response to cytoreductive therapy. Finally, digital PCR was more sensitive in detecting KIT D816V in bone marrow sections of indolent systemic mastocytosis patients than melting curve analysis after peptide nucleic acid-mediated PCR clamping (97% vs 89%; P<0.05). In summary, digital PCR-based measurement of KIT D816V mutation burden in the tissue represents a novel biomarker with independent prognostic significance that can also be employed for monitoring disease progression and treatment response in systemic mastocytosis.

Comparison of BCR-ABL1 quantification in peripheral blood and bone marrow using an International Scale-standardized assay for assessment of deep molecular response in chronic myeloid leukemia.

Background Monitoring of molecular response (MR) using quantitative polymerase chain reaction (PCR) for BCR-ABL1 is a pivotal tool for guiding tyrosine kinase inhibitor therapy and the long-term follow-up of patients with chronic myeloid leukemia (CML). Results of MR monitoring are standardized according to the International Scale (IS), and specific time-dependent molecular milestones for definition of optimal response and treatment failure have been included in treatment recommendations. The common practice to use peripheral blood (PB) instead of bone marrow (BM) aspirate to monitor the MR monitoring in CML has been questioned. Some studies described differences between BCR-ABL1 levels in paired PB and BM specimens. Methods We examined 631 paired PB and BM samples from 283 CML patients in a retrospective single-center study using an IS normalized quantitative reverse transcription (qRT)-PCR assay for quantification of BCR-ABL1IS. Results A good overall concordance of BCR-ABL1IS results was found, a systematic tendency towards higher BCR-ABL1IS levels in PB was observed in samples of CML patients in a major MR. This difference was most pronounced in patients treated with imatinib for at least 1 year. Importantly, the difference resulted in a significantly lower rate of deep MR when BCR-ABL1IS was assessed in the PB compared to BM aspirates. Conclusions In summary, our data suggest that the classification of deep MR in patients with CML is more stringent in PB than in BM. Our study supports the current practice to primarily use PB for long-term molecular follow-up monitoring in CML.

Digital PCR: A Sensitive and Precise Method for KIT D816V Quantification in Mastocytosis.

BACKGROUND: The analytically sensitive detection of KIT D816V in blood and bone marrow is important for diagnosing systemic mastocytosis (SM). Additionally, precise quantification of the KIT D816V variant allele fraction (VAF) is relevant clinically because it helps to predict multilineage involvement and prognosis in cases of advanced SM. Digital PCR (dPCR) is a promising new method for sensitive detection and accurate quantification of somatic mutations. METHODS: We performed a validation study of dPCR for KIT D816V on 302 peripheral blood and bone marrow samples from 156 patients with mastocytosis for comparison with melting curve analysis after peptide nucleic acid-mediated PCR clamping (clamp-PCR) and allele-specific quantitative real-time PCR (qPCR). RESULTS: dPCR showed a limit of detection of 0.01% VAF with a mean CV of 8.5% and identified the mutation in 90% of patients compared with 70% for clamp-PCR (P < 0.001). Moreover, dPCR for KIT D816V was highly concordant with qPCR without systematic deviation of results, and confirmed the clinical value of KIT D816V VAF measurements. Thus, patients with advanced SM showed a significantly higher KIT D816V VAF (median, 2.43%) compared with patients with indolent SM (median, 0.14%; P < 0.001). Moreover, dPCR confirmed the prognostic significance of a high KIT D816V VAF regarding survival (P < 0.001). CONCLUSIONS: dPCR for KIT D816V provides a high degree of precision and sensitivity combined with the potential for interlaboratory standardization, which is crucial for the implementation of KIT D816V allele burden measurement. Thus, dPCR is suitable as a new method for KIT D816V testing in patients with mastocytosis.

Two Novel Missense Mutations and a 5bp Deletion in the Erythroid-Specific Promoter of the PKLR Gene in Two Unrelated Patients With Pyruvate Kinase Deficient Transfusion-Dependent Chronic Nonspherocytic Hemolytic Anemia.

We report two children with severe chronic hemolytic anemia, the cause of which was difficult to establish because of transfusion dependency. Reduced erythrocyte pyruvate kinase activity in their asymptomatic parents provided the diagnostic clues for mutation screening of the PKLR gene and revealed that one child was a compound heterozygote of a novel paternally derived 5-bp deletion in the promoter region (c.-88_-84delTCTCT) and a maternally derived missense mutation in exon nine (c.1174G>A; p.Ala392Thr). The second child was a compound heterozygote of two novel missense mutations, namely a paternally derived exon ten c.1381G>A (p.Glu461Lys) and a maternally derived exon seven c.907-908delCC (p.Pro303GlyfsX12) variant.

Effect of preanalytical time-delay on platelet function as measured by multiplate, PFA-100 and VerifyNow.

BACKGROUND AND AIMS: Platelet function testing may help to identify poor responders to antiplatelet drugs. The aim of this study was to compare three commonly used platelet function tests with special focus on the pre-analytical influence of time-delay on the tested parameters. METHODS: We assessed ADP-induced platelet function by the Multiplate, Platelet Function Analyzer-100 (PFA-100) and VerifyNow in nine healthy volunteers and 36 patients receiving clopidogrel or prasugrel 1 and 3 hours after sampling. RESULTS: The PFA-100 demonstrated non-closure time in 23 patients. A more graded response could be detected with the two other devices. Aggregation in whole blood (Multiplate) decreased after 3 hours compared to 1 hour in all subjects (p < 0.05). Furthermore, aggregation levels obtained by the VerifyNow showed a decrease in patients taking P2Y12 inhibitors after 3 hours (p < 0.05), except in three patients, in whom an increase was observed. CONCLUSION: Responses to ADP are time-dependent after blood sampling for the Multiplate in all subjects and for the VerifyNow in patients on antiplatelet drugs. For both devices, platelet aggregation was reduced 3 hours after sampling which may affect data interpretation and clinical consequences.

Sepsis biomarkers in neutropaenic systemic inflammatory response syndrome patients on standard care wards.

BACKGROUND: Neutropaenic patients are at a high risk of contracting severe infections. In particular, in these patients, parameters with a high negative predictive value are desirable for excluding infection or bacteraemia. This study evaluated sepsis biomarkers in neutropaenic patients suffering from systemic inflammatory response syndrome (SIRS). Further, the predictive capacities of evaluated biomarkers in neutropaenic SIRS patients were compared to non-neutropaenic SIRS patients. MATERIAL AND METHODS: In this prospective observational cohort study, patients with clinically suspected sepsis were screened. The predictive capacities of procalcitonin (PCT), C-reactive protein and lipopolysaccharide-binding protein (LBP) in neutropaenic SIRS patients were evaluated in terms of their potential to identify infection or bacteraemia and were compared to results for non-neutropaenic SIRS patients. To select an appropriate control cohort, propensity score matching was applied, balancing confounding factors between neutropaenic and non-neutropaenic SIRS patients. RESULTS: Of 3370 prospectively screened patients with suspected infection, 51 patients suffered from neutropaenic SIRS. For the identification of infection, none of the assessed biomarkers presented a clinically relevant discriminatory potency. Lipopolysaccharide-binding protein and PCT demonstrated discriminatory capacity to discriminate between nonbacteraemic and bacteraemic SIRS in patients with neutropaenia [receiver-operating characteristics-area under the curves (ROC-AUCs): 0.860, 0.818]. In neutropaenic SIRS patients, LBP had a significantly better ROC-AUC than in a comparable non-neutropaenic patient cohort for identifying bacteraemia (P = 0.01). CONCLUSION: In neutropaenic SIRS patients, none of the evaluated biomarkers was able to adequately identify infection. LBP and PCT presented a good performance in identifying bacteraemia. Therefore, these markers could be used for screening purposes to increase the pretest probability of blood culture analysis.

Sepsis in standard care: patients' characteristics, effectiveness of antimicrobial therapy and patient outcome--a cohort study.

PURPOSE: Fast diagnosis and initiation of appropriate antibiotic therapy is pivotal for the survival of sepsis patients. However, most studies on suspected sepsis patients are conducted in the intensive care unit or in the emergency room setting, neglecting the standard care setting. This study evaluated sepsis risk factors, microbiological accurateness of the initial empiric antimicrobial therapy and its effect on hospital mortality in standard care patients. METHODS: In this prospective observational cohort study, patients with clinically suspected sepsis meeting two or more SIRS criteria were screened on standard care wards. After hospital discharge, occurrence of an infection was assessed according to standardized criteria, and empirical antibiotic therapy was evaluated using antibiograms of recognized pathogens by expert review. RESULTS: Of the 2384 screened patients, 298 fulfilled two or more SIRS criteria. Among these were 28.2 % SIRS patients without infection, 46.3 % non-bacteremic/fungemic sepsis patients and 25.5 % bacteremic/fungemic sepsis patients. Occurrence of a malignant disease and chills were associated with a higher risk of patients having bacteremic/fungemic sepsis, whereas other described risk factors remained insignificant. In total, 91.1 % of suspected sepsis patients received empirical antimicrobial therapy, but 41.1 % of bacteremic sepsis patients received inappropriate therapy. Non-surviving bacteremic sepsis patients received a higher proportion of inappropriate therapy than those who survived (p = 0.022). CONCLUSIONS: A significant proportion of bacteremic sepsis patients receive inappropriate empiric antimicrobial therapy. Our results indicate that rapid availability of microbiological results is vital, since inappropriate antimicrobial therapy tended to increase the hospital mortality of sepsis patients.

Evaluation of the automated coagulation analyzer CS-5100 and its utility in high throughput laboratories.

BACKGROUND: Automated analyzers are an important component of modern laboratories. As a representative of the newest generation of coagulation analyzers, the CS-5100 features several technical refinements including a pre-analytical assessment unit as well as multi-wavelength optical detection units. Therefore, the CS-5100 is supposed to rapidly and accurately perform a broad panel of coagulation tests. In the current study, the CS-5100 was evaluated regarding its precision and practicability in a clinical laboratory setting. METHODS: The CS-5100 was evaluated regarding its intra- and inter-assay precision using commercially available control samples. RESULTS of patient samples, including hemolytic, icteric and lipemic specimens, measured on the CS-5100 were compared to reference analyzers, which are used in our accredited laboratory. RESULTS: The coefficients of variation, assessed in the intra- and inter-assay precision analyses were below 5% representatively for most parameters. RESULTS, obtained by the CS-5100 showed predominantly a high comparability to used reference analyzers, with correlation coefficients ranging from 0.857 to 0.990. Only minor ranged systemic or proportional differences were found in Passing-Bablok regression between the CS-5100 and reference analyzers regarding most of the tested parameters. Lipemic samples had a tendency to deteriorate correlation coefficients, but an overall effect of the sample's triglyceride level could be ruled out. In a routine setting, the analyzer reached a sample throughput rate of 160 tests per hour. CONCLUSIONS: The CS-5100 is able to rapidly and precisely measure patient samples. No considerable influence on test comparability was found for elevated levels of free hemoglobin, bilirubin or triglycerides.

Performance Evaluation of the Fully Automated NeuMoDx RT-PCR Platform for the Quantification of CMV and EBV DNA in EDTA Plasma: Implications for Clinical Management and Establishment of a Conversion Formula.

The NeuMoDx96 platform is a fully automated real-time PCR (RT-PCR) system. To provide continued testing quality with the introduction of new assays, the primary aim of this study was to evaluate the analytical and clinical performance of the NeuMoDx platform for the detection and quantification of CMV and EBV DNA in EDTA plasma. As no conversion from log10 international units per milliliter to copies per milliliter was provided, the secondary aim was to calculate and establish a conversion factor for the output of results in copies per milliliter for CMV and EBV. Archived ETDA plasma samples (cytomegalovirus [CMV], n = 290; Ebstein-Barr virus [EBV], n = 254) were used to evaluate the analytical performance of the NeuMoDx96 platform against the routine real-time quantitative PCR (qPCR) assays. Additionally, the first WHO international standards (WHO-IS) for CMV (n = 70) and EBV (n = 72) were used for the calculation of the intra- and interassay variation. WHO-IS qualitative agreement between the assays was 100%. Intra-assay variability was low for both CMV assays (coefficient of variation [CV], phosphate-buffered saline [PBS], 3 log10 IU/mL NeuMoDx, 3.67%; Abbott RealTime, CMV, 3.35%) and NeuMoDx EBV assay (CV, PBS, 3 log10 IU/mL, 3.05%) but high for the Altona EBV assay (CV, PBS, 3 log10 IU/mL, 26.13%). The overall qualitative concordance in clinical samples was 96.8% (270/279) for CMV and 96.7% (237/245) for EBV. The mean difference between the assays was -0.2 log10 IU/mL (CMV) and -0.18 log10 IU/mL (EBV). High qualitative concordance and a significant correlation of quantitative values for both assays make NeuMoDx CMV and EBV assays suitable for routine diagnostic testing. The new RT-PCR system and conversion formulas to report results in copies per milliliter are now applied in clinical routine testing. IMPORTANCE Clinical management of solid organ transplant (SOT) patients requires the careful monitoring of immunosuppression and viral infection or reactivation. qPCR is the gold standard for the detection and quantification of very small amounts of viral DNA and allows for an early assessment of viral load kinetics. The tested NeuMoDx 96 platform provides faster results than the previously used RT-PCR workflows for CMV (Abbott m2000 and RealTime CMV assay) and EBV (LightCycler 480 II, Roche high pure extraction, and Altona RealStar EBV assay) DNA detection. The implemented conversion formulas allow the continued reporting in clinically established copies per milliliter, important for long-term care of SOT patients.

Biomarkers for differentiation of coronavirus disease 2019 or extracorporeal membrane oxygenation related inflammation and bacterial/fungal infections in critically ill patients: A prospective observational study.

Secondary infections in coronavirus disease 2019 (COVID-19) patients are difficult to distinguish from inflammation associated with COVID-19 and/or extracorporeal membrane oxygenation (ECMO). Therefore, highly specific and sensitive biomarkers are needed to identify patients in whom antimicrobial therapy can be safely withheld. In this prospective monocentric study, 66 COVID-19 patients admitted to the intensive care unit (ICU) for ECMO evaluation were included. A total of 46 (70%) patients with secondary infections were identified by using broad microbiological and virological panels and standardized diagnostic criteria. Various laboratory parameters including C-reactive protein (CRP), interleukin (IL)-6, procalcitonin (PCT), and IL-10 were determined at time of study inclusion. The best test performance for differentiating bacterial/fungal secondary infections and COVID-19 and/or ECMO associated inflammation was achieved by IL-10 (ROC-AUC 0.84) and a multivariant step-wise regression model including CRP, IL-6, PCT, and IL-10 (ROC-AUC 0.93). Data obtained in the present study highlights the use of IL-10 to differentiate secondary bacterial/fungal infections from COVID-19 and/or ECMO associated inflammation in severely ill COVID-19 patients.

One-Year Follow-Up of COVID-19 Patients Indicates Substantial Assay-Dependent Differences in the Kinetics of SARS-CoV-2 Antibodies.

Determination of antibody levels against the nucleocapsid (N) and spike (S) proteins of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are used to estimate the humoral immune response after SARS-CoV-2 infection or vaccination. Differences in the design and specification of antibody assays challenge the interpretation of test results, and comparative studies are often limited to single time points per patient. We determined the longitudinal kinetics of antibody levels of 145 unvaccinated coronavirus disease 2019 (COVID-19) patients at four visits over 1 year upon convalescence using 8 commercial SARS-CoV-2 antibody assays (from Abbott, DiaSorin, Roche, Siemens, and Technoclone), as well as a virus neutralization test (VNT). A linear regression model was used to investigate whether antibody results obtained in the first 6 months after disease onset could predict the VNT results at 12 months. Spike protein-specific antibody tests showed good correlation to the VNT at individual time points (rS, 0.74 to 0.92). While longitudinal assay comparison with the Roche Elecsys anti-SARS-CoV-2 S test showed almost constant antibody concentrations over 12 months, the VNT and all other tests indicated a decline in serum antibody levels (median decrease to 14% to 36% of baseline). The antibody level at 3 months was the best predictor of the VNT results at 12 months after disease onset. The current standardization to a WHO calibrator for normalization to binding antibody units (BAU) is not sufficient for the harmonization of SARS-CoV-2 antibody tests. Assay-specific differences in absolute values and trends over time need to be considered when interpreting the course of antibody levels in patients. IMPORTANCE Determination of antibodies against SARS-CoV-2 will play an important role in detecting a sufficient immune response. Although all the manufacturers expressed antibody levels in binding antibody units per milliliter, thus suggesting comparable results, we found discrepant behavior between the eight investigated assays when we followed the antibody levels in a cohort of 145 convalescent patients over 1 year. While one assay yielded constant antibody levels, the others showed decreasing antibody levels to a varying extent. Therefore, the comparability of the assays must be improved regarding the long-term kinetics of antibody levels. This is a prerequisite for establishing reliable antibody level cutoffs for sufficient individual protection against SARS-CoV-2.

Different Types of Coagulase Are Associated With 28-Day Mortality in Patients With Staphylococcus aureus Bloodstream Infections.

Background:Staphylococcus aureus (S. aureus), a leading cause of bacteremia and infective endocarditis, exploits the human coagulation system by using a wide range of specific virulence factors. However, the impact of these host-pathogen interactions on the outcome of patients with Staphylococcus aureus bacteremia (SAB) remains unclear. Methods: A total of 178 patients with S. aureus bacteremia were included and analyzed regarding bacterial factors (coa gene size, vWbp, clfA, clfB, fnbA, fnbB, fib) and clinical parameters. A stepwise multivariate Cox regression model and a Partitioning Around Medoids (PAM) cluster algorithm were used for statistical analysis. Results: Patients' risk factors for 28-day mortality were creatinine (OR 1.49, p < 0.001), age (OR 1.9, p < 0.002), fibrinogen (OR 0.44, p < 0.004), albumin (OR 0.63, p < 0.02), hemoglobin (OR 0.59, p < 0.03), and CRP (OR 1.72, p < 0.04). Five distinct bacterial clusters with different mortality rates were unveiled, whereof two showed a 2-fold increased mortality and an accumulation of specific coagulase gene sizes, 547-base pairs and 660-base pairs. Conclusions: Based on the data obtained in the present study an association of coagulase gene size and fib regarding 28-day mortality was observed in patients with S. aureus bloodstream infections. Further animal and prospective clinical studies are needed to confirm our preliminary findings.

The Role of Iron and Erythropoietin in the Association of Fibroblast Growth Factor 23 with Anemia in Chronic Kidney Disease in Humans.

Anemia in chronic kidney disease (CKD) is an almost universal complication of this condition. Fibroblast growth factor 23 (FGF23), a key-player in mineral metabolism, is reportedly associated with anemia and hemoglobin levels in non-dialysis CKD patients. Here, we sought to further characterize this association while taking into account the biologically active, intact fraction of FGF23, iron metabolism, and erythropoietin (EPO). Hemoglobin, EPO, iron, and mineral metabolism parameters, including both intact and c-terminal-FGF23 (iFGF23 and cFGF23, respectively) were measured cross-sectionally in 225 non-dialysis CKD patients (stage 1-5, median eGFR: 30 mL/min./1.73m2) not on erythropoiesis stimulating agents or intravenous iron therapy. Statistical analysis was performed by multiple linear regression. After adjustment for eGFR and other important confounders, only cFGF23 but not iFGF23 was significantly associated with hemoglobin levels and this association was largely accounted for by iron metabolism parameters. cFGF23 but not iFGF23 was also associated with mean corpuscular hemoglobin (MCH) and mean corpuscular volume (MCV), again in dependence on iron metabolism parameters. Similarly, EPO concentrations were associated with cFGF23 but not iFGF23, but their contribution to the association of cFGF23 with hemoglobin levels was marginal. In pre-dialysis CKD patients, the observed association of FGF23 with hemoglobin seems to be restricted to cFGF23 and largely explained by the iron status.

Human diamine oxidase is readily released from activated neutrophils ex vivo and in vivo but is rarely elevated in bacteremic patients.

During human diamine oxidase (DAO) ELISA development we noticed that in serum DAO concentrations appear to be higher when compared to plasma. Neutrophils contain DAO in the specific granules and we hypothesized that DAO is released from neutrophils during serum coagulation. If activation of neutrophils can release DAO, its concentrations might be elevated in vivo after lipopolysaccharide (LPS) administration and in bacteremic patients. Using blood from healthy volunteers DAO concentrations were measured ex vivo in serum, citrate, EDTA and heparin plasma over several hours and after activation of neutrophils. Lipopolysaccharide and granulocyte-colony stimulating factor (G-CSF) were administered to 15 and 8 healthy volunteers, respectively and DAO concentrations were measured at different timepoints. DAO antigen levels were also determined in three different subcohorts of patients with culture-proven bacteremia and high C-reactive protein (CRP) levels. DAO concentrations were elevated in a time-dependent manner in serum but not in EDTA or citrate plasma (P < 0.01). Neutrophil activation using phorbol myristate acetate (PMA) and zymosan dose-dependently caused DAO concentrations to be elevated more than 10-fold at both 22°C and 37°C (both P-values <0.001). Administration of LPS to healthy volunteers released DAO from neutrophils (P < 0.001). Of the 55 different bacteremic patients selected from three independent cohorts only 3 (5.4%) showed highly elevated DAO concentrations. Serum DAO concentrations do not accurately reflect circulating enzyme levels but coagulation-induced neutrophil activation and consequently DAO release. Only a few bacteremic patients show high DAO concentrations able to degrade histamine rapidly.

Development of a fully automated high throughput PCR for the detection of SARS-CoV-2: The need for speed.

Currently, testing for coronavirus is performed with time and personnel consuming PCR assays. The aim of this study was to evaluate the sensitivity, specificity and capacity of a fully automated, random access high-throughput real-time PCR-based diagnostic platform for the detection of SARS-CoV-2. The NeuMoDx N96 system displayed an equal or better detection rate for SARS-CoV-2 compared with the LightCycler 480II system and showed a specificity of 100%. The median PCR run time for all 28 PCR runs was 91 (IQR 84-97) minutes. The capacity of the NeuMoDx N96 could easily surpass the capacity of most currently used molecular test systems and significantly reduce the turn-around time.