Short-term biological variation of serum tryptase.

OBJECTIVES: Serum tryptase is a biomarker of mast cell activation. Among others, it is used in the diagnosis of anaphylaxis where a significant increase during the acute phase supports the diagnosis. When evaluating changes in biomarker levels, it is of utmost importance to consider the biological variation of the marker. Therefore, the aim of this study was to evaluate the short-term biological variation of serum tryptase. METHODS: Blood samples were drawn at 9 AM three days in a row from apparently healthy subjects. On day two, additional blood samples were drawn every third hour for 12 h. The tryptase concentration was measured in serum using a fluoroenzyme immunoassay (ImmunoCAP™, Thermo Fisher Scientific). Linear mixed-effects models were used to calculate components of biological variation. RESULTS: In 32 subjects, the overall mean concentration of tryptase was 4.0 ng/mL (range, 1.3-8.0 ng/mL). The within-subject variation was 3.7 % (95 % confidence interval (CI) 3.0-4.4 %), the between-subject variation was 31.5 % (95 % CI 23.1-39.8 %), and the analytical variation was 3.4 % (95 % CI 2.9-4.1 %). The reference change value was 13.3 % for an increase in tryptase at a 95 % level of significance. No significant day-to-day variation was observed (p=0.77), while a minute decrease in the serum concentration was observed during the day (p<0.0001). CONCLUSIONS: Serum tryptase is a tightly regulated biomarker with very low within-subject variation, no significant day-to-day variation, and only minor semidiurnal variation. In contrast, a considerable between-subject variation exists. This establishes serum tryptase as a well-suited biomarker for monitoring.

Reference intervals for platelet large cell ratio, platelet distribution width, plateletcrit and standard haematological parameters determined on the Sysmex XN-10 in a cohort of 30,917 Danish blood donors.

With the introduction of the Sysmex XN-10, new platelet indices reflecting platelet maturity can be obtained alongside with a full blood count. Therefore, the need for verified reference intervals is present. The purpose of this study was to establish reference intervals for the platelet indices: platelet large cell ratio (P-LCR), platelet distribution width (PDW) and plateletcrit (PCT) in a large Scandinavian cohort. Furthermore, we aimed to verify previously established reference intervals of haematological parameters included in a full blood count. Blood samples were obtained from healthy Danish blood donors and analysed by use of the Sysmex XN-10 analyser (Sysmex, Kobe, Japan). Non-parametric 95% reference intervals were determined as the 2.5 and 97.5 percentile and presented with 90% confidence intervals (CI). In total, 30,917 blood donors were included and the following reference intervals were established: P-LCR, ratio: 17.3 (90% CI: 17.2 - 17.5) - 46.2 (90% CI:45.9 - 46.4); P DW, fL:9.5 (90% CI: 9.5 - 9.5) - 17.2 (90% CI: 17.2 - 17.3) and PCT, fraction: 0.18 (0.18 - 0.18) - 0.38 (0.38 - 0.39). The reference intervals were stable across age and sex. Furthermore, reference intervals for the remaining haematological parameters included in a full blood count were verified and found in line with the previously established reference intervals.

Influence of age and sex on osmoscan indies for next-generation osmotic gradient ektacytometry.

BACKGROUND AND AIMS: Osmotic gradient ektacytometry is an important method for diagnosis of red blood cell membrane disorders. For interpretation of the osmoscan parameters on the ektacytomety, an age-matched control sample drawn at the same time is recommended for direct comparison. However, this can be challenging for laboratories to fulfil, especially when ektacytometry is performed in children. Therefore, the aim of this study was to evaluate the influence of age and sex on the osmoscan parameters. MATERIALS AND METHODS: Blood samples from 231 subjects were analyses on a LoRRca MaxSIS. Data were investigated for need of partitioning by age and sex. After outlier detection, reference intervals (RIs) for osmoscan parameters were estimated. RESULTS: For all parameters except EImin, lower values were observed in infants < 3 month (N = 50) than in all other age group. Hence, RIs were calculated separately for this age group. For EImin, a unified RI was calculated. No difference between sexes was observed for any of the parameters. CONCLUSION: Lower RIs and a left shift in the osmoscan curves were observed in infants < 3 months compared with older subjects. Hence, age-matched controls are necessary when evaluating ektacytometry in newborns, but can be ignored in older children and adults. This will ease the laboratory workflow when performing ektacytometry.

Pediatric reference intervals of the hemoglobin fractions HbA2, HbF and HbA0 using high-performance liquid chromatography and capillary electrophoresis.

INTRODUCTION: ß-thalassemia is a common genetic disorder with an estimated prevalence of 80-90 million carriers worldwide. As elevated hemoglobin A2 (HbA2) is a primary feature of carriers, hemoglobin fraction analysis is a common technique used for initial screening. However, pediatric reference intervals (RIs) are scarce. Hence, the aim was to establish pediatric RIs of hemoglobin fractions using high-performance liquid chromatography (HPLC) and capillary electrophoresis (CE). METHODS: Samples were collected from assumed healthy children and adolescents of 1-18 years. Analyses were conducted using the Tosoh Automated Glycohemoglobin Analyzer HLC-723®G11 (Tosoh G11, HPLC) and the Capillarys 3 Octa (CE). Data were investigated for need of partitioning by both age (1-6 years vs. 6-18 years) and sex. RESULTS: In total, 189 and 196 subjects were included in the statistical analysis of HPLC and CE, respectively. The 95% RI of HbA2 was 2.00-2.90% by HPLC and 2.2-3.0% by CE. Partitioning of data was not clinically relevant by HPLC. However, partitioning by age was suggested by CE. CONCLUSION: RIs of hemoglobin fractions in individuals of 1-18 years using commercially available HPLC and CE equipment were reported. This is the first report of a pediatric RI of HbA2 using the Tosoh G11.

Extracellular 2'-5' oligoadenylate synthetase stimulates RNase L-independent antiviral activity: a novel mechanism of virus-induced innate immunity.

The 2'-5' oligoadenylate synthetase (OAS) proteins are traditionally considered intracellular antiviral proteins. However, several studies demonstrate a correlation between the concentration of freely circulating OAS protein in sera from hepatitis C patients and their clinical prognosis. Here we demonstrate that extracellular OAS1 enters into cells and possesses a strong antiviral activity, both in vitro and in vivo, which is independent of RNase L. The OAS protein directly inhibits viral proliferation and does not require the activation of known antiviral signaling pathways. We propose that OAS produced by cells infected with viruses is released to the extracellular space, where it acts as a paracrine antiviral agent. Thus, the OAS protein represents the first direct antiviral compound released by virus-infected cells.

Rapid Uptake and Inhibition of Viral Propagation by Extracellular OAS1.

The oligoadenylate synthetase (OAS) proteins are traditionally considered intracellular antiviral proteins that mediate antiviral activity through the synthesis of 2'-5'-linked oligoadenylates and subsequent activation of the endoribonuclease RNase L. However, we have recently demonstrated that exogenous recombinant OAS1 is taken up by cells and reduces viral replication both in cell culture and in vivo, independent of RNase L. These results demonstrate a novel paracrine antiviral activity of OAS working in parallel with the classical RNase L pathway. In this study, we investigate the uptake kinetics of recombinant porcine OAS1 and show that it is rapidly and efficiently internalized in a manner that can be blocked by heparin. Heparin, furthermore, abolishes the antiviral activity of OAS1, demonstrating the requirement of the intracellular localization of OAS1 to inhibit the virus. In addition, we demonstrate that exogenous OAS1 affects an early step of the viral replication cycle.

The oligoadenylate synthetase family: an ancient protein family with multiple antiviral activities.

The 2'-5' oligoadenylate synthetases (OAS) are interferon-induced antiviral enzymes that recognize virally produced dsRNA and initiate RNA destabilization through activation of RNase L within infected cells. However, recent evidence points toward several RNase L-independent pathways, through which members of the OAS family can exert antiviral activity. The crystal structure of OAS led to a novel insight into the catalytic mechanism, and revealed a remarkable similarity between OAS, Polyadenosine polymerase, and the class I CCA-adding enzyme from Archeoglobus fulgidus. This, combined with a variety of bioinformatic data, leads to the definition of a superfamily of template independent polymerases and proved that the OAS family are ancient proteins, which probably arose as early as the beginning of metazoan evolution.

Differential regulation of the OASL and OAS1 genes in response to viral infections.

The 2'-5' oligoadenylate synthetase (OAS) family consist of three genes encoding active OAS enzymes (OAS1-3) and an OAS-Like (OASL) gene encoding an inactive protein. The transcription of all four members of this family is actively induced by interferon (IFN), but so far no attempt to systematically analyze the expression of these genes during viral infection has been made. We analyzed the expression of the human OAS1 and OASL genes in response to infection with Sendai virus or Influenza A virus. Surprisingly, we found a marked difference in the expression pattern of these genes. Our data showed that the OASL gene is rapidly induced in response to viral infection and that this induction is mediated by IFN regulatory factor 3 (IRF-3). In contrast to the OASL gene, the induction of the OAS1 gene by virus infection was lower, and did require a functional type I IFN response. The pronounced difference in gene regulation between the OAS1 and OASL genes agrees with a functional difference between these genes, which must exist as a consequence of the lack of the 2-5A synthetase activity of the OASL protein. Furthermore, the behavior of the OASL gene is consistent with the behavior of an antiviral gene.