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Background Recent data propose a diagnostic and prognostic capacity for citrullinated histone H3 (H3Cit), a marker of neutrophil extracellular traps (NETs), in pathologic conditions such as cancer and thrombosis. However, current research is hampered by lack of standardized assays. Objectives We aimed to develop an assay to reliably quantify nucleosomal H3Cit in human plasma. Methods We assessed the common practice of in vitro enzymatically modified histone H3 as calibration standards and the specificity of available intrapeptidyl citrulline antibodies. Based on our findings, we developed and validated a novel assay to quantify nucleosomal H3Cit in human plasma. Results We show that enzymatically citrullinated H3 proteins are compromised by high enzyme-dependent lot variability as well as instability in plasma. We furthermore demonstrate that the majority of commercially available antibodies against intrapeptidyl citrulline display poor specificity for their reported target when tested against a panel of semi-synthetic nucleosomes containing distinct histone H3 citrullinations. Finally, we present a novel assay utilizing highly specific monoclonal antibodies and semi-synthetic nucleosomes containing citrulline in place of arginine at histone H3, arginine residues 2, 8, and 17 (H3R2,8,17Cit) as calibration standards. Rigorous validation of this assay shows its capacity to accurately and reliably quantify nucleosomal H3Cit levels in human plasma with clear elevations in cancer patients compared to healthy individuals. Conclusions Our novel approach using defined nucleosome controls enables reliable quantification of H3Cit in human plasma. This assay will be broadly applicable to study the role of histone citrullination in disease and its utility as a biomarker.

There is an emerging interest in the diverse functions of neutrophil extracellular traps (NETs) in a variety of disease settings. However, data on circulating NETs rely largely upon surrogate NET markers such as cell-free DNA, nucleosomes, and NET-associated enzymes. Citrullination of histone H3 by peptidyl arginine deiminase 4 (PAD4) is central for NET formation, and citrullinated histone H3 (H3Cit) is considered a NET-specific biomarker. We therefore aimed to optimize and validate a new enzyme-linked immunosorbent assay (ELISA) to quantify the levels of H3Cit in human plasma. A standard curve made of in vitro PAD4-citrullinated histones H3 allows for the quantification of H3Cit in plasma using an anti-histone antibody as capture antibody and an anti-histone H3 citrulline antibody for detection. The assay was evaluated for linearity, stability, specificity, and precision on plasma samples obtained from a human model of inflammation before and after lipopolysaccharide injection. The results revealed linearity and high specificity demonstrated by the inability of detecting non-citrullinated histone H3. Coefficients of variation for intra- and inter-assay variability ranged from 2.1 to 5.1% and from 5.8 to 13.5%, respectively, allowing for a high precision. Furthermore, our results support an inflammatory induction of a systemic NET burden by showing, for the first time, clear intra-individual elevations of plasma H3Cit in a human model of lipopolysaccharide-induced inflammation. Taken together, our work demonstrates the development of a new method for the quantification of H3Cit by ELISA that can reliably be used for the detection of NETs in human plasma.

Citrullinated histone H3 (H3Cit) is a central player in the neutrophil release of nuclear chromatin, known as neutrophil extracellular traps (NETs). NETs have been shown to elicit harmful effects on the host, and were recently proposed to promote tumor progression and spread. Here we report significant elevations of plasma H3Cit in patients with advanced cancer compared with age-matched healthy individuals. These elevations were specific to cancer patients as no increase was observed in severely ill and hospitalized patients with a higher non-malignant comorbidity. The analysis of neutrophils from cancer patients showed a higher proportion of neutrophils positive for intracellular H3Cit compared to severely ill patients. Moreover, the presence of plasma H3Cit in cancer patients strongly correlated with neutrophil activation markers neutrophil elastase (NE) and myeloperoxidase (MPO), and the inflammatory cytokines interleukin-6 and -8, known to induce NETosis. In addition, we show that high levels of circulating H3Cit strongly predicted poor clinical outcome in our cohort of cancer patients with a 2-fold increased risk for short-term mortality. Our results also corroborate the association of NE, interleukin-6 and -8 with poor clinical outcome. Taken together, our results are the first to unveil H3Cit as a potential diagnostic and prognostic blood marker associated with an exacerbated inflammatory response in patients with advanced cancer.

Sample fixation is an important issue in flow cytometric platelet assays. However, previous reports were less than consistent regarding the influence of sample fixation on the assays. We evaluated the effects of formaldehyde and paraformaldehyde fixation on platelet P-selectin expression and fibrinogen binding using whole-blood flow cytometry and a Coulter EPICS XL-MCL cytometer. Fluorescent-labeled whole-blood samples were diluted with HEPES-buffered saline or fixed with formaldehyde (0.2, 0.5, and 1. 0%) or paraformaldehyde (0.5, 1.0, and 2.0%). Platelet P-selectin expression was 1.1+/-0.3% and 39.6+/-13.7% in unfixed resting and 10(-5) M ADP stimulated samples, respectively. Resting P-selectin expression was not significantly altered by 0.2 or 0.5% formaldehyde fixation, but was slightly decreased by 1.0% formaldehyde fixation or PFA fixation. Formaldehyde fixation caused small increases of P-selectin expression in ADP-stimulated samples. Compared to platelet fibrinogen binding of unfixed resting (4.5+/-2.1%) and ADP-stimulated (56.7+/-22.6%) samples, formaldehyde or paraformaldehyde fixation had no significant influence on resting samples, but mildly increased fibrinogen binding in stimulated samples. Unfixed samples were stable for 2 h. Fixed samples were generally stable for at least 6 h, but not thereafter. Thus, formaldehyde and paraformaldehyde have mild but complex influences on platelet P-selectin expression and fibrinogen binding measurements. To evaluate the stabilities of unfixed and fixed samples, samples were analyzed after different durations (0, 1, 2, 4, 6, 12, and 24 h) of storage at 4 degrees C in the dark. The results suggest that sample manipulation without fixation may be used when the samples are analyzed within 2 h, and that fixation with 0.5-1.0% formaldehyde or paraformaldehyde seems to be preferable when sample analysis is delayed. Effects of fixation should be carefully evaluated when establishing flow cytometric platelet assays in every laboratory.

8 subjects were exposed to the Stroop mental performance test in a design with alternating hourly periods of rest and stress. During each period one urine sample and several venous plasma samples were obtained. Heart rate responded rapidly to initiation and termination of the stress exposure with increases and decreases respectively. Both urinary and plasma adrenaline increased significantly during stress. The plasma response was immediate and sustained. Neither urinary, nor plasma noradrenaline were significantly increased by the test. Plasma noradrenaline, however, increased significantly on termination of the exposure to stress. It was suggested that the latter effect may be due to muscle sympathetic nerve activity decreasing during stress and increasing following stress. The sample-to-sample variation was more than 20% of the mean for both catecholamines, indicating the need for frequent sampling to reliably reflect plasma levels. The mean intraindividual plasma/urine correlation was r = 0.70 (p less than 0.001) for adrenaline and r = 0.40 (p less than 0.05) for noradrenaline. When only resting periods were considered, no significant correlations remained, apparently due to a reduced range of variation and accompanying reduced signal-to-noise ratio. It is concluded that both urinary and plasma adrenaline may be useful in the evaluation of changes in sympatho-adrenal activity during stress.

1. 1. A comparison is made between the sympatho-adrenal regulation of subcutaneous adipose tissue blood flow in the dog and in man. 2. 2. In the dog neuronally released noradrenaline causes vasoconstriction in subcutaneous adipose tissue by preferential activation of α-adrenoceptors located close to the sympathetic nerve terminals. 3. 3. In man sympathetic reflex activation also causes vasoconstriction in subcutaneous adipose tissue, presumably via activation of α-adrenoceptors. 4. 4. The adipose tissue vascular β-adrenoceptors are of the β1-subtype in the dog and the β2-subtype in man. 5. 5. Because of this adrenaline causes vasoconstriction in dog adipose tissue and vasodilation in human adipose tissue.

High performance liquid chromatography with electrochemical detection has been compared to a radioenzymatic method for the determination of plasma catecholamines. With the use of an internal standard highly accurate determinations of plasma noradrenaline, adrenaline and dopamine were performed on 0.2–2 ml plasma with the chromatographic method. The radioenzymatic method required only 3 × 50 μl plasma. A comparison of noradrenaline and adrenaline concentrations measured by the two methods in a set of nine plasma samples showed an excellent agreement between the methods (r=0.993 and 0.994, respectively). Advantages and disadvantages with the two methods are discussed.