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Background: In neonates, hypoglycemia is an emergency condition requiring urgent treatment. Therefore, rapid and reliable blood glucose measurements are necessary. However, this step has been proven difficult because of both analytical and preanalytical variables. In our children's hospital, we incidentally observed cases of hypoglycemia that were not in line with the clinical picture of the infants. Remarkably, most of these infants had a high hematocrit., Methods: Glucose concentrations were determined in blood samples from healthy participants that were collected in Li-heparin capillary and pediatric tubes. The effect of hematocrit on glucose consumption over time was studied by artificially increasing sample hematocrits. To study the effect of sample cooling, glucose concentrations were followed over time in samples stored at room temperature and on ice., Results: In all samples, glucose concentrations declined with time. This effect was most dramatic [up to 18 mg/dL (1 mmol/L) in the first 30 min] in samples with high hematocrits and collected in capillary tubes. Cooling of samples clearly reduced glucose consumption; however, this was not evident in the first 30 min., Conclusions: Overestimation of hypoglycemia in infants must be considered if samples are not centrifuged or are not analyzed immediately after sampling. The extent of overestimation is more pronounced in samples with a high hematocrit, collected in capillary tubes. Cooling of samples does not prevent glucose consumption in vitro during the first 30 min. These results emphasize that, for glucose analysis, prompt handling of samples of newborns with a high hematocrit is necessary., (© 2016 by American Association for Clinical Chemistry.)

Background: Point-of-care D-dimer tests have recently been introduced to enable rapid exclusion of deep venous thrombosis (DVT) without the need to refer a patient for conventional laboratory-based D-dimer testing. Before implementation in practice, however, the diagnostic accuracy of each test should be validated., Methods: We analyzed data of 577 prospectively identified consecutive primary care patients suspected to have DVT, who underwent 5 point-of-care D-dimer tests-4 quantitative (Vidas®, Pathfast™, Cardiac®, and Triage®) and 1 qualitative (Clearview Simplify®)-and ultrasonography as the reference method. We evaluated the tests for the accuracy of their measurements and submitted a questionnaire to 20 users to assess the user-friendliness of each test., Results: All D-dimer tests showed negative predictive values higher than 98%. Sensitivity was high for all point-of-care tests, with a range of 0.91 (Clearview Simplify) to 0.99 (Vidas). Specificity varied between 0.39 (Pathfast) and 0.64 (Clearview Simplify). The quantitative point-of-care tests showed similar and high discriminative power for DVT, according to calculated areas under the ROC curves (range 0.88-0.89). The quantitative Vidas and Pathfast devices showed limited user-friendliness for primary care, owing to a laborious calibration process and long analyzer warm-up time compared to the Cardiac and Triage. For the qualitative Clearview Simplify assay, no analyzer or calibration was needed, but interpretation of a test result was sometimes difficult because of poor color contrast., Conclusions: Point-of-care D-dimer assays show good and similar diagnostic accuracy. The quantitative Cardiac and Triage and the qualitative Clearview Simplify D-dimer seem most user-friendly for excluding DVT in the doctor's office.

Objective: Macrophage activation syndrome (MAS) in systemic onset juvenile idiopathic arthritis (SoJIA) is considered to be an acquired form of familial haemophagocytic lymphohistiocytosis (fHLH). FHLH is an autosomal recessive disorder, characterized by diminished NK cell function and caused by mutations in the perforin gene (PRF1) in 20-50% of patients. Interestingly, SoJIA patients display decreased levels of perforin in NK cells and diminished NK cell function as well. Here, we analysed PRF1 and its putative promoter in SoJIA patients with or without a history of MAS., Methods: DNA of 56 SoJIA patients (41 Italian and 15 Dutch) was isolated. Of these, 15 (27%) had a confirmed history of MAS. We sequenced PRF1 and 1.5 kb of the 5'-upstream region. DNA sequence variations in the promoter region were functionally tested in transfection experiments using a human NK cell line., Results: We detected a previously undescribed sequence variation (-499 C > T) in the promoter of PRF1 in 18% of the SoJIA patients. However, transfection experiments did not show functional implications of this variation. Secondly, we found that 11 of 56 (20%) SoJIA patients were heterozygous for missense mutations in PRF1. In particular, we found a high prevalence of the Ala91Val mutation, a variant known to result in defective function of perforin. Interestingly, the prevalence of Ala91Val in SoJIA patients with a history of MAS (20%) was increased compared with SoJIA patients without MAS (9.8%). One SoJIA patient, heterozygous for Ala91Val, showed profound decreased perforin levels at the time of MAS., Conclusions: These findings suggest that PRF1 mutations play a role in the development of MAS in SoJIA patients.

Background: Although promoter mutations are known to cause functionally important consequences for gene expression, promoter analysis is not a regular part of DNA diagnostics., Content: This review covers different important aspects of promoter mutation analysis and includes a proposed model procedure for studying promoter mutations. Characterization of a promoter sequence variation includes a comprehensive study of the literature and databases of human mutations and transcription factors. Phylogenetic footprinting is also used to evaluate the putative importance of the promoter region of interest. This in silico analysis is, in general, followed by in vitro functional assays, of which transient and stable transfection assays are considered the gold-standard methods. Electrophoretic mobility shift and supershift assays are used to identify trans-acting proteins that putatively interact with the promoter region of interest. Finally, chromatin immunoprecipitation assays are essential to confirm in vivo binding of these proteins to the promoter., Summary: Although promoter mutation analysis is complex, often laborious, and difficult to perform, it is an essential part of the diagnosis of disease-causing promoter mutations and improves our understanding of the role of transcriptional regulation in human disease. We recommend that routine laboratories and research groups specialized in gene promoter research cooperate to expand general knowledge and diagnosis of gene-promoter defects.