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Unlabelled: We report on four children with heparin-induced thrombocytopenia type II. In three patients, therapy with unfractionated heparin was associated with development of cardiac thrombi or with thrombosis progression up to the inferior vena cava or with aggravation of peripheral arterial occlusion. In the fourth child, the disease was recognized early on, and no complication occurred. Heparin-induced thrombocytopenia type II was confirmed by heparin-induced platelet activation assay and/or heparin/platelet factor 4-ELISA. Concomitant elevated antiphospholipid antibodies were seen in all patients. Danaparoid sodium applied at a dosage of between 1.2 and 7.1 U/kg/h stopped the disease progression in each patient. Three children had a clinical recovery with partial recanalization, but for the child with peripheral arterial occlusion disease, amputation of some of the toes became necessary.Conclusion: Our data indicate that heparin-induced thrombocytopenia type II is a potential life-threatening disease in children and danaparoid sodium is beneficial in this age group. [ABSTRACT FROM AUTHOR]

In recent years, instrumental improvements have enabled the spread of mass spectrometry-based lipidomics platforms in biomedical research. In mass spectrometry, the reliability of generated data varies for each compound, contingent on, among other factors, the availability of labeled internal standards. It is challenging to evaluate the data for lipids without specific labeled internal standards, especially when dozens to hundreds of lipids are measured simultaneously. Thus, evaluation of the performance of these platforms at the individual lipid level in interlaboratory studies is generally not feasible in a time-effective manner. Herein, using a focused subset of sphingolipids, we present an in-house validation methodology for individual lipid reliability assessment, tailored to the statistical analysis to be applied. Moreover, this approach enables the evaluation of various methodological aspects, including discerning coelutions sharing identical selected reaction monitoring transitions, pinpointing optimal labeled internal standards and their concentrations, and evaluating different extraction techniques. While the full validation according to analytical guidelines for all lipids included in a lipidomics method is currently not possible, this process shows areas to focus on for subsequent method development iterations as well as the robustness of data generated across diverse methodologies., (© 2024. The Author(s).)

Plasma extracellular vesicle (EV) number and composition are altered following myocardial infarction (MI), but to properly understand the significance of these changes it is essential to appreciate how the different isolation methods affect EV characteristics, proteome and sphingolipidome. Here, we compared plasma EV isolated from platelet-poor plasma from four healthy donors and six MI patients at presentation and 1-month post-MI using ultracentrifugation (UC), polyethylene glycol precipitation, acoustic trapping, size-exclusion chromatography (SEC) and immunoaffinity capture. The isolated EV were evaluated by Nanoparticle Tracking Analysis (NTA), Western blot, transmission electron microscopy (TEM), an EV-protein array, untargeted proteomics (LC-MS/MS) and targeted sphingolipidomics (LC-MS/MS). The application of the five different plasma EV isolation methods in patients presenting with MI showed that the choice of plasma EV isolation method influenced the ability to distinguish elevations in plasma EV concentration following MI, enrichment of EV-cargo (EV-proteins and sphingolipidomics) and associations with the size of the infarct determined by cardiac magnetic resonance imaging 6 months post-MI. Despite the selection bias imposed by each method, a core of EV-associated proteins and lipids was detectable using all approaches. However, this study highlights how each isolation method comes with its own idiosyncrasies and makes the comparison of data acquired by different techniques in clinical studies problematic., Competing Interests: The Author(s) declare that there is no conflict of interest., (© 2022 The Authors. Journal of Extracellular Biology published by Wiley Periodicals, LLC on behalf of the International Society for Extracellular Vesicles.)

Octadecanoids are broadly defined as oxylipins ( i.e., lipid mediators) derived from 18-carbon fatty acids. In contrast to the well-studied eicosanoids, there is a lack of analytical methods for octadecanoids, hampering further investigations in the field. We developed an integrated workflow combining chiral separation by supercritical fluid chromatography (SFC) and reversed-phase liquid chromatography (LC) coupled to tandem mass spectrometry detection for quantification of a broad panel of octadecanoids. The platform includes 70 custom-synthesized analytical and internal standards to extend the coverage of the octadecanoid synthetic pathways. A total of 103 octadecanoids could be separated by chiral SFC and complex enantioseparations could be performed in <13 min, while the achiral LC method separated 67 octadecanoids in 13.5 min. The LC method provided a robust complementary approach with greater sensitivity relative to the SFC method. Both methods were validated in solvent and surrogate matrix in terms of linearity, lower limits of quantification (LLOQ), recovery, accuracy, precision, and matrix effects. Instrumental linearity was good for both methods ( R 2 > 0.995) and LLOQ ranged from 0.03 to 6.00 ng/mL for SFC and 0.01 to 1.25 ng/mL for LC. The average accuracy in the solvent and surrogate matrix ranged from 89 to 109% in SFC and from 106 to 220% in LC, whereas coefficients of variation (CV) were <14% (at medium and high concentrations) and 26% (at low concentrations). Validation in the surrogate matrix showed negligible matrix effects (<16% for all analytes), and average recoveries ranged from 71 to 83%. The combined methods provide a platform to investigate the biological activity of octadecanoids and expand our understanding of these little-studied compounds.

Apolipoprotein E (apoE) occurs on the majority of plasma lipoproteins and plays a major role in the lipid metabolism in the periphery and in the central nervous system. ApoE is a polymorphic protein with three common isoforms, apoE2, apoE3 and apoE4, derived from respective alleles ε2, ε3 and ε4. The aim of this study was to develop a sample pretreatment protocol combined with rapid mass spectrometry (MS)-based assay for simultaneous apolipoprotein profiling and apoE phenotype identification. This assay was validated in 481 samples from patients with stable atherosclerotic cardiovascular disease (ASCVD) and applied to study association with mild cognitive impairment (MCI) in the LIFE Adult study, including overall 690 study subjects. Simultaneous quantification of 8−12 major apolipoproteins including apoA-I, apoB-100 and apoE could be performed within 6.5 min. Phenotyping determined with the developed MS assay had good agreement with the genotyping by real-time fluorescence PCR (97.5%). ApoE2 isoform was associated with the highest total apoE concentration compared to apoE3 and apoE4 (p < 0.001). In the subgroup of diabetic atherosclerotic cardiovascular disease (ASCVD) patients, apoE2 isoform was related to higher apoC-I levels (apoE2 vs. apoE3, p < 0.05), while in the subgroup of ASCVD patients under statin therapy apoE2 was related to lower apoB-100 levels (apoE2 vs. apoE3/apoE4, p < 0.05). A significant difference in apoE concentration observed between mild cognitive impairment (MCI) subjects and controls was confirmed for each apoE phenotype. In conclusion, this study provides evidence for the successful implementation of an MS-based apoE phenotyping assay, which can be used to assess phenotype effects on plasma lipid and apolipoprotein levels.

In the late 1980s an HIV-1 epidemic emerged in Romania that was dominated by subtype F1. The main route of infection is believed to be parenteral transmission in children. We sequenced partial pol coding regions of 70 subtype F1 samples from children and adolescents from the PENTA-EPPICC network of which 67 were from Romania. Phylogenetic reconstruction using the sequences and other publically available global subtype F sequences showed that 79% of Romanian F1 sequences formed a statistically robust monophyletic cluster. The monophyletic cluster was epidemiologically linked to parenteral transmission in children. Coalescent-based analysis dated the origins of the parenteral epidemic to 1983 [1981-1987; 95% HPD]. The analysis also shows that the epidemic's effective population size has remained fairly constant since the early 1990s suggesting limited onward spread of the virus within the population. Furthermore, phylogeographic analysis suggests that the root location of the parenteral epidemic was Bucharest.

Background: Tick-borne encephalitis in children appears to be more benign than in adults and shows also a more favourable outcome. Only some authors report of sequelae like paralysis, paresis or seizures and behavioural abnormalities. The aim was to describe the clinical features of tick-borne encephalitis in children with special attention to sequelae and to review the literature., Methods: Retrospective review of all charts of children with serologically confirmed tick-borne encephalitis hospitalised in Styria between 1981 and 2005., Results: One hundred sixteen children were diagnosed with tick-borne encephalitis. Ninety-two children (79.3%) developed meningitis and 24 (20.7%) meningoencephalitis. Eleven patients with meningoencephalitis showed somnolence, 5 confusion, 5 tremors, 2 facial palsy, 1 ataxia, 1 epileptic seizure and 1 hemi paresis. Seven patients had to be admitted to the intensive care unit. Two children (1.7%) developed long time neurological sequelae: one epileptic seizure with requirement of antiepileptic therapy and one left-sided hemi paresis. One hundred twelve children had been not and 3 incompletely vaccinated against tick-borne encephalitis. Only one child had been fully vaccinated according to the Austrian vaccination schedule., Conclusion: Our study provides further evidence that tick-borne encephalitis in children has a substantial morbidity and in single cases severe long-time neurological sequelae are observed.

Background: An association has been described between mortality in children with meningococcal disease and functional polymorphisms in the interleukin-1 (IL1) cluster. We undertook a multicenter study to evaluate associations of these polymorphisms in a Central European population., Patients and Methods: The study involved 95 Middle European pediatric hospitals. We collected blood samples from, and clinical information about, 285 previously healthy children with meningococcal infection. We used a newly developed multiplexed mutagenic separated PCR assay to analyze 6 polymorphisms within the IL1 cluster: IL1A (-889)C/T, IL1A (+4845)G/T, IL1B (-511)C/T, IL1B (-31)C/T, IL1B (+3954), and IL1RA (+2018)C/T. We studied the same polymorphisms in a comparison group of 481 healthy newborns., Results: Genotype frequencies between patients and the comparison group differed significantly only for the IL1RA (+2018)C/T variant: The CC genotype was more frequent in patients (11%) than in healthy controls (5%; P = 0.008). In the patient group, the C allele was significantly more prevalent (67%) in nonsurvivors than in survivors (42%; P = 0.02)., Conclusion: The IL1RA (+2018)C/T polymorphism is associated with the risk of meningococcal disease and with its outcome.

Unlabelled: Meningococcal disease may present as sepsis, meningitis or a combination of both. Impaired fibrinolysis and massive elevation of the plasminogen activator inhibitor-1 (PAI-1) is a characteristic feature of meningococcal sepsis. Previously, an association between mortality and the functional 4G/5G promoter polymorphism of the PAI-1gene in a cohort of UK and Dutch children with meningococcal sepsis was reported. We carried out a prospective, multicentre study to investigate the association of the 4G/5G PAI-1 polymorphism, diagnosis, and outcome in meningococcal disease in a Central European and UK population. Blood samples and clinical information of 347 previously healthy children with meningococcal infection were collected from 95 paediatric hospitals in Germany, Switzerland, Italy, the United Kingdom, and Austria from 2000 until 2002. Mortality was significantly associated with the 4G/4G genotype (12 of 90 (13%) vs. 15 of 240 (6%), P = 0.037), resulting in an odds ratio of 2.31. The diagnosis of sepsis (independent of symptoms of meningitis) was significantly more frequent in carriers of the 4G/4G genotype (P = 0.01), resulting in an odds ratio of 2.21 to develop sepsis. Meningitis was not associated with the PAI-1 4G/5G polymorphism, and allele frequencies were similar in patient and control groups., Conclusion: Our data show a correlation between the 4G/4G genotype in the plasminogen activator inhibitor-1 gene and poor outcome in children with meningococcal infection. In addition, 4G homozygous patients were prone to develop sepsis. We found no influence of the plasminogen activator inhibitor-1 polymorphism on the susceptibility to invasive meningococcal infection.

Sleeping disorders in general practice are common, but as the main reason for seeking help they only account for 1% of all consultations in all age groups. The aim of our study was to find out the overall frequency and consulting patterns for sleeping disorders in patients (over sixty years old) in general practices in eastern Austria. In this age group, sleeping disorders accounted for 7% of all reasons for seeking consultation. This percentage increased to 45% if the patients were asked if they suffered from insomnia. Half of the patients reported nycturia, but not every patient interpreted this occurrence as a real sleeping disorder. In accordance with the literature, we found a high prevalence of sleeping disorders in the unselected elderly patients visiting the surgery for highly different reasons.

Unlabelled: We report on four children with heparin-induced thrombocytopenia type II. In three patients, therapy with unfractionated heparin was associated with development of cardiac thrombi or with thrombosis progression up to the inferior vena cava or with aggravation of peripheral arterial occlusion. In the fourth child, the disease was recognized early on, and no complication occurred. Heparin-induced thrombocytopenia type II was confirmed by heparin-induced platelet activation assay and/or heparin/platelet factor 4-ELISA. Concomitant elevated antiphospholipid antibodies were seen in all patients. Danaparoid sodium applied at a dosage of between 1.2 and 7.1 U/kg/h stopped the disease progression in each patient. Three children had a clinical recovery with partial recanalization, but for the child with peripheral arterial occlusion disease, amputation of some of the toes became necessary., Conclusion: Our data indicate that heparin-induced thrombocytopenia type II is a potential life-threatening disease in children and danaparoid sodium is beneficial in this age group.

If specialists expect general practitioners to carry out extensive investigations serving early detection of diseases they don't consider how limited in that respect the possibilities of general practitioners are. In general, practice however the early detection of malignancies is satisfactory if the doctor makes use of the short time available the best possible.

Background: Burning pain of red and warm hands and/or feet are the classical symptoms of erythromelalgia., Case Report: We describe the symptoms of acute idiopathic erythromelalgia and arterial hypertension in a five-year-old boy. Five days after a gastroenteritis the patient developed burning hands and feet in combination with arterial hypertension. Typically continuous cooling of all affected limbs was necessary to relieve the pain. Drug therapy with sodium nitroprusside only relieved the pain and dropped the blood pressure temporarily. Five weeks after onset of the disease all symptoms disappeared and the patient is still free of complaints (follow up period: 2 years). In the view of the presented case we discuss the differential diagnoses as well as therapeutical options.