Elevated NET, Calprotectin, and Neopterin Levels Discriminate between Disease Activity in COVID-19, as Evidenced by Need for Hospitalization among Patients in Northern Italy.

Coronavirus disease 2019 (COVID-19) displays clinical heterogeneity, but little information is available for patients with mild or very early disease. We aimed to characterize biomarkers that are useful for discriminating the hospitalization risk in a COVID-19 cohort from Northern Italy during the first pandemic wave. We enrolled and followed for four weeks 76 symptomatic SARS-CoV-2 positive patients and age/sex-matched healthy controls. Patients with mild disease were discharged (n.42), and the remaining patients were hospitalized (n.34). Blood was collected before any anti-inflammatory/immunosuppressive therapy and assessed for soluble C5b-9/C5a, H3-neutrophil extracellular traps (NETs), calprotectin, and DNase plasma levels via ELISA and a panel of proinflammatory cytokines via ELLA. Calprotectin and NET levels discriminate between hospitalized and non-hospitalized patients, while DNase negatively correlates with NET levels; there are positive correlations between calprotectin and both NET and neopterin levels. Neopterin levels increase in patients at the beginning of the disease and do so more in hospitalized than non-hospitalized patients. C5a and sC5b-9, and other acute phase proteins, correlate with neopterin, calprotectin, and DNase. Both NET and neopterin levels negatively correlate with platelet count. We show that calprotectin, NETs, and neopterin are important proinflammatory parameters potentially useful for discriminating between COVID-19 patients at risk of hospitalization.

DNase analysed by a novel competitive assay in patients with complications after ChAdOx1 nCoV-19 vaccination and in normal unvaccinated blood donors.

Increased levels of neutrophil extracellular traps (NETs) have been detected in individuals with vaccine complications after the ChAdOx1 nCov vaccine with a correlation between the severity of vaccine side effects and the level of NETosis. DNases may disrupt NETs by degrading their content of DNA, and a balance has been reported between NETs and DNases. Because of this and since the inflammatory marker NETs may be used as a confirmatory test in diagnosing VITT, it is of interest to monitor levels of DNase in patients with increased NETs levels. The current novel rapid DNase ELISA was tested in blood samples of patients with known increased levels of NETs with or without VITT after ChAdOx1 nCoV-19 vaccination. DNase levels in VITT patients were significantly increased compared with normal unvaccinated blood donors and compared with patients with post-vaccination symptoms but not VITT. However, since EDTA was found to inhibit DNase, serum and not EDTA-plasma samples should be applied for DNase testing. The novel DNase assay may serve as a supplementary test to the NETs test when analysing samples from patients with suspected increased NETs levels.

Inflammatory markers calprotectin, NETs, syndecan-1 and neopterin in COVID-19 convalescent blood donors.

Persisting inflammation has been discovered in lungs and other parenchymatous organs of some COVID-19 convalescents. Calprotectin, neutrophil extracellular traps (NETs), syndecan-1 and neopterin are general key inflammatory markers, and systemically enhanced levels of them may remain after the COVID-19 infection. These inflammatory markers were therefore measured in serum samples of 129 COVID-19 convalescent and 27 healthy blood donors or employees at Oslo Blood bank, Norway. Also antibodies against SARS-CoV-2 nucleocapsid antigen were measured, and timing of sampling and severity of infection noted. Whereas neopterin and NETs values remained low and those for syndecan-1 were not raised to statistically significant level, concentrations for calprotectin, as measured by a novel mixed monoclonal assay, were significantly increased in the convalescents. Antibodies against SARS-CoV-2 nucleocapsid antigen were elevated, but did not correlate with levels of inflammatory markers. Difference between the groups in only one biomarker makes evaluation of ongoing or residual inflammation in the convalescents difficult. If there is a low-grade inflammation, it would in that case involve neutrophils.

Elevated NETs and Calprotectin Levels after ChAdOx1 nCoV-19 Vaccination Correlate with the Severity of Side Effects.

ChAdOx1 nCoV-19 vaccination has been associated with the rare side effect; vaccine-induced immune thrombotic thrombocytopenia (VITT). The mechanism of thrombosis in VITT is associated with high levels of neutrophil extracellular traps (NETs). The present study examines whether key markers for NETosis, such as H3-NETs and calprotectin, as well as syndecan-1 for endotheliopathy, can be used as prognostic factors to predict the severity of complications associated with ChAdOx1 vaccination. Five patients with VITT, 10 with prolonged symptoms and cutaneous hemorrhages but without VITT, and 15 with only brief and mild symptoms after the vaccination were examined. Levels of H3-NETs and calprotectin in the vaccinated individuals were markedly increased in VITT patients compared to vaccinees with milder vaccination-associated symptoms, and a strong correlation (r ≥ 0.745, p < 0.001) was found with severity of vaccination side effects. Syndecan-1 levels were also positively correlated (r = 0.590, p < 0.001) in vaccinees to side effects after ChAdOx1 nCoV-19 vaccination. We hypothesize that the inflammatory markers NETs and calprotectin may be used as confirmatory tests in diagnosing VITT.

NETs analysed by novel calprotectin-based assays in blood donors and patients with multiple myeloma or rheumatoid arthritis: A pilot study.

Two novel enzyme-linked immunosorbent assays (ELISAs), designed to detect complexes containing DNA, leucocyte calprotectin and S100A12 proteins, were generated for improved specificity and rapid measurement of neutrophil extracellular traps (NETs). The assays were applied on plasma and serum samples from blood donors for establishment of reference values, and from patients with multiple myeloma (MM) or rheumatoid arthritis (RA) in order to examine putatively increased values in the two different inflammatory conditions. Although NETs were hardly detectable in healthy individuals, NET levels were as expected highly and statistically significantly increased in RA patients. The detection of statistically significantly increased NET levels in MM is a novel finding.

The effect of pre-storage cooling on 2,3-DPG levels in red cells stored in SAG-M.

BACKGROUND: The concentration of red cell 2,3-DPG (2,3-diphosphoglycerate) rapidly decreases during storage. A favourable effect on red cell 2,3-DPG has been demonstrated by rapid cooling of whole blood prior to storage. In our study we have investigated how different methods of cooling whole blood immediately after donation effect 2,3-DPG levels during storage. STUDY DESIGN AND METHODS: Thirty-six whole blood units (in 6 groups) of 450 ml were collected in 63 ml CPD. SAG-M was used as preservative solution for red cell concentrates (RCC). The units in one group were cooled down at ambient temperature, while units in the other groups were cooled down rapidly by different ways immediately after bleeding. Samples from the whole blood units were collected at various days during storage for 2,3-DPG measurements. RESULTS: The decline in 2,3-DPG during the first two weeks of storage was significantly slower in the groups which were cooled down rapidly to 17-18 degrees C within 1h after bleeding (all p

Calprotectin plasma level is elevated in preeclampsia.

BACKGROUND: Calprotectin is a protein found in myelomonocytic cells and plays a role in various physiological functions such as inflammatory processes and antiproliferation of cells, and in the neutrophil defense against bacterial infections. Preeclampsia is characterized by maternal endothelial dysfunction and by insufficient trophoblast invasion into the maternal endometrium (decidua). In addition, preeclampsia is associated with maternal leukocyte activation and we therefore wanted to investigate whether calprotectin levels in plasma from women with preeclampsia differed from the levels in normotensive pregnant and nonpregnant women. METHOD: Calprotectin measurements were included in a case-control study of 20 preeclamptic women matched with 20 normotensive pregnant women regarding age, pregnancy length, parity and body mass index (BMI). We also measured calprotectin in 12 nonpregnant women. Calprotectin plasma levels were analyzed using an enzyme-linked immunosorbent assay (ELISA). RESULTS: We discovered significantly elevated plasma calprotectin levels in preeclamptic patients compared to matched normotensive pregnancies: 768 (612-1016) microg/L vs. 445 (276-598) microg/L (medians, 25, 75 percentiles, respectively), p = 0.002. CONCLUSIONS: The elevated plasma calprotectin levels demonstrated in the preeclampsia group supports the notion that leukocytes are activated in preeclampsia. The elevated calprotectin level might constitute a part of the innate defense in myelomonocytic cells against microorganisms in pregnancy. We suggest further elucidation of a role for calprotectin in the development of pregnancy disorders such as preeclampsia.