Comparison of five Anti-SARS-CoV-2 antibody assays across three doses of BNT162b2 reveals insufficient standardization of SARS-CoV-2 serology.

OBJECTIVES: To investigate the comparability of WHO standard referenced commercial SARS-CoV-2 antibody tests over three doses of BNT162b2 vaccine and up to 14 months. METHODS: 114 subjects (without previous SARS-CoV-2 infection or immunosuppressive medication) vaccinated with three doses of BNT162b2 were included in this study. Antibody levels were quantified 3 weeks after the first dose, 5-6 weeks and 7 months after the second dose, and 4-5 weeks and 4 months after the third dose using the Roche Elecsys SARS-CoV-2 S, the Abbott SARS-CoV-2 IgG II Quant, the DiaSorin LIAISON SARS-CoV-2 TrimericS IgG, the GenScript cPASS sVNT and the TECO sVNT assays. RESULTS: For each time point analyzed, systematic differences are evident between the results in BAU/mL of the three antibody binding assays. The assay ratios change in a time-dependent manner even beyond administering the third dose (Roche measuring 9 and 3 times higher than Abbott and DiaSorin, respectively). However, changes decrease in magnitude with increasing time intervals from the first dose. IgG-based assays show better agreement across them than with Roche (overall correlations: Abbott x DiaSorin: ρ = 0.94 vs. Abbott x Roche: ρ=0.89, p < 0.0001; DiaSorin x Roche: ρ = 0.87, p < 0.0001), but results are not interchangeable. The sVNTs suggest an underestimation of antibody levels by Roche and slight overestimation by both IgG assays after the first vaccine dose. CONCLUSIONS: Standardization of SARS-CoV-2 antibody binding assays still needs to be improved to allow reliable use of variable assay systems for longitudinal analyses.

Increasing test specificity without impairing sensitivity: lessons learned from SARS-CoV-2 serology.

BACKGROUND: Serological tests are widely used in various medical disciplines for diagnostic and monitoring purposes. Unfortunately, the sensitivity and specificity of test systems are often poor, leaving room for false-positive and false-negative results. However, conventional methods were used to increase specificity and decrease sensitivity and vice versa. Using SARS-CoV-2 serology as an example, we propose here a novel testing strategy: the 'sensitivity improved two-test' or 'SIT²' algorithm. METHODS: SIT² involves confirmatory retesting of samples with results falling in a predefined retesting zone of an initial screening test, with adjusted cut-offs to increase sensitivity. We verified and compared the performance of SIT² to single tests and orthogonal testing (OTA) in an Austrian cohort (1117 negative, 64 post-COVID-positive samples) and validated the algorithm in an independent British cohort (976 negatives and 536 positives). RESULTS: The specificity of SIT² was superior to single tests and non-inferior to OTA. The sensitivity was maintained or even improved using SIT² when compared with single tests or OTA. SIT² allowed correct identification of infected individuals even when a live virus neutralisation assay could not detect antibodies. Compared with single testing or OTA, SIT² significantly reduced total test errors to 0.46% (0.24-0.65) or 1.60% (0.94-2.38) at both 5% or 20% seroprevalence. CONCLUSION: For SARS-CoV-2 serology, SIT² proved to be the best diagnostic choice at both 5% and 20% seroprevalence in all tested scenarios. It is an easy to apply algorithm and can potentially be helpful for the serology of other infectious diseases.

Preanalytical stability of SARS-CoV-2 anti-nucleocapsid antibodies.

OBJECTIVES: Anti-nucleocapsid (NC) antibodies are produced in response to SARS-CoV-2 infection. Therefore, they are well suited for the detection of a previous infection. Especially in the case of seroprevalence studies or during the evaluation of a novel in-vitro diagnostic test, samples have been stored at <-70 °C (short- and long-term) or 2-10 °C (short-term) before analysis. This study aimed to assess the impact of different storage conditions relevant to routine biobanking on anti-NC antibodies. METHODS: The preanalytical impact of short-term storage (84 [58-98] days) on <-70 °C and for 14 days at 2-10 °C was evaluated using samples from 111 donors of the MedUni Vienna Biobank. Long-term effects (443 [409-468] days) were assessed using 208 samples from Biobank Graz and 49 samples from Biobank Vienna. Anti-Nucleocapsid antibodies were measured employing electrochemiluminescence assays (Roche Anti-SARS-CoV-2). RESULTS: After short-term storage, the observed changes did not exceed the extent that could be explained by analytical variability. In contrast, results after long-term storage were approximately 20% higher and seemed to increase with storage duration. This effect was independent of the biobank from which the samples were obtained. Accordingly, the sensitivity increased from 92.6 to 95.3% (p=0.008). However, comparisons with data from Anti-Spike protein assays, where these deviations were not apparent, suggest that this deviation could also be explained by the analytical variability of the qualitative Anti-NC assay. CONCLUSIONS: Results from anti-NC antibodies are stable during short-term storage at <-70 °C and 2-10 °C. After long-term storage, a slight increase in sensitivity could not be ruled out.

The Comparability of Anti-Spike SARS-CoV-2 Antibody Tests is Time-Dependent: a Prospective Observational Study.

Various commercial anti-Spike SARS-CoV-2 antibody tests are used for studies and in clinical settings after vaccination. An international standard for SARS-CoV-2 antibodies has been established to achieve comparability of such tests, allowing conversions to BAU/mL. This study aimed to investigate the comparability of antibody tests regarding the timing of blood collection after vaccination. For this prospective observational study, antibody levels of 50 participants with homologous AZD1222 vaccination were evaluated at 3 and 11 weeks after the first dose and 3 weeks after the second dose using two commercial anti-Spike binding antibody assays (Roche and Abbott) and a surrogate neutralization assay. The correlation between Roche and Abbott changed significantly depending on the time point studied. Although Abbott provided values three times higher than Roche 3 weeks after the first dose, the values for Roche were twice as high as for Abbott 11 weeks after the first dose and 5 to 6 times higher at 3 weeks after the second dose. The comparability of quantitative anti-Spike SARS-CoV-2 antibody tests was highly dependent on the timing of blood collection after vaccination. Therefore, standardization of the timing of blood collection might be necessary for the comparability of different quantitative SARS-COV-2 antibody assays. IMPORTANCE This work showed that the comparability of apparently standardized SARS-CoV-2 antibody assays (Roche, Abbott; both given in BAU/mL) after vaccination depends on the time of blood withdrawal. Initially (3 weeks after the first dose AZD1222), there were 3 times higher values in the Abbott assay, but this relationship inversed before boosting (11 weeks after the first dose) with Roche 2 times greater than Abbott. After the booster, Roche quantified ca. 5 times higher levels than Abbott. This must be considered by clinicians when interpreting SARS-CoV-2 antibody levels.

Serum antibody response to BNT162b2 after natural SARS-CoV-2 infection.

BACKGROUND: There is preliminary evidence that individuals with previous SARS-CoV-2 infections exhibit a more pronounced antibody response. However, these assumptions have not yet been supported by data obtained through various CE-marked tests. This study aimed to close this gap. METHODS: Sixty-nine seronegatives and 12 individuals post-SARS-CoV-2 infection (tested by CE-labelled Roche NC immunoassay or PCR-confirmed assay) were included 21 ± 1 days after receiving the first dose of the Pfizer/BioNTech BNT162b2 vaccine. Antibody response to viral spike protein (S) was assessed by CE-labelled Roche S and DiaSorin S1/S2 assays and by a surrogate virus neutralization test (sVNT). RESULTS: After a single dose of BNT162b2, individuals after natural SARS-CoV-2 infection presented with markedly higher anti-S levels than naïve individuals (Roche S: 9078.5 BAU/mL [5267.0-24 298.5] vs 79.6 [24.7-142.3]; and DiaSorin S1/S2: 1465.0 AU/mL [631.0-5365.0] vs 63.7 [47.8-87.5]) and showed all the maximum observed inhibition activity in the sVNT (98%), without overlaps between groups. There was a trend for higher responses in those with a more distant infection, although not statistically significant. The relative antibody increase after dose 2 was significantly higher among naïve individuals (25-fold), but antibody levels remained below that of seropositives. CONCLUSIONS: Compared with naïve individuals, seropositives after natural SARS-CoV-2 infection presented with a substantially higher antibody response already after dose 1 of BNT162b2, as measured by two CE-marked in vitro diagnostic tests and a sVNT. These results should stimulate discussion and research on whether individuals after previous SARS-CoV-2 infection would benefit from a two-part vaccination schedule or whether these currently much-needed second doses could be saved.

Initial SARS-CoV-2 vaccination response can predict booster response for BNT162b2 but not for AZD1222.

OBJECTIVE: To determine whether severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) antibody levels after the first dose of vaccine can predict the final antibody response, and whether this is dependent on the vaccine type. METHODS: Sixty-nine recipients of BNT162b2 (Pfizer/BioNTech) and 55 recipients of AZD1222 (AstraZeneca), without previous infection or immunosuppressive medication, were included in this study. Antibody levels were quantified 3 weeks after the first dose [directly before boostering in the case of AZD1222 (11 weeks after the first dose)] and 3 weeks after the second dose using the Roche Elecsys SARS-CoV-2 S total antibody assay. RESULTS: Median pre-booster {BNT162b2: 80.6 [interquartile range (IQR) 25.5-167.0] binding antibody units (BAU)/mL; AZD1222: 56.4 (IQR 36.4-104.8) BAU/mL; not significant} and post-booster [BNT162b2: 2092.0 (IQR 1216.3-4431.8) BAU/mL; AZD1222: 957.0 (IQR 684.5-1684.8) BAU/mL; P<0.0001] levels correlated well in the recipients of BNT162b2 (ρ=0.53) but not in the recipients of AZD1222. Moreover, antibody levels after the first dose of BNT162b2 correlated inversely with age (ρ=-0.33, P=0.013), whereas a positive correlation with age was observed after the second dose in recipients of AZD1222 (ρ=0.26, P=0.030). CONCLUSIONS: The results of this study suggest that antibody levels quantified by the Roche Elecsys SARS-CoV-2 S assay before the booster shot could infer post-booster responses to BNT162b2, but not to AZ1222. In addition, this study found a vaccine-dependent effect on antibody responses, where age seems to play an ambivalent role.

Anti-Spike Protein Assays to Determine SARS-CoV-2 Antibody Levels: a Head-to-Head Comparison of Five Quantitative Assays.

Reliable quantification of the antibody response to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is highly relevant, e.g., for identifying possible vaccine failure and estimating the time of protection. Therefore, we evaluated five different anti-SARS-CoV-2 antibody assays regarding the quantification of anti-spike (S) antibodies. Sera from 69 SARS-CoV-2-naive individuals 21 ± 1 days after vaccination with a single dose of BNT162b2 (Pfizer/BioNTech) were tested using the following quantitative assays: Roche S total antibody, DiaSorin trimeric spike IgG, DiaSorin S1/S2 IgG, Abbott II IgG, and Serion/Virion IgG. Results were further compared to the percent inhibition calculated from a surrogate virus neutralization test (sVNT). Individual values were distributed over several orders of magnitude for all assays. Although the assays were in good overall agreement (ρ = 0.80 to 0.94), Passing-Bablok regression revealed systematic constant and proportional differences, which could not be eliminated by converting the results to binding antibody units (BAU) per milliliter, as suggested by the manufacturers. Seven (10%) individuals had negative sVNT results (i.e., <30% inhibition). These samples were identified by most assays and yielded significantly lower binding antibody levels. Although all assays showed good correlation, they were not interchangeable, even when converted to BAU per milliliter using the WHO international standard for SARS-CoV-2 immunoglobulin. This highlights the need for further standardization of SARS-CoV-2 serology. IMPORTANCE Reliable quantification of the antibody response to SARS-CoV-2 is highly relevant, e.g., for identifying possible vaccine failure and estimating the time of protection. We compared the performance of five CE marked tests that quantify antibodies against the viral spike protein. Our findings suggest that, although all assays showed good correlation, their results were not interchangeable, even when converted to BAU per milliliter using the WHO international standard for SARS-CoV-2 immunoglobulin. This highlights the need for further standardization of SARS-CoV-2 serology.

Basal myokine levels are associated with quality of life and depressed mood in older adults.

In an aging society, late-life depression has become an increasing problem. There is evidence that physical activity ameliorates depressive symptoms and increases the quality of life (QoL). However, the underlying mechanisms are still poorly understood. Myokines are molecules secreted in response to muscle contraction. Some of them can cross the blood-brain barrier, making them promising candidates for mediating the beneficial effects of physical activity on mood. The present study aims to compare circulating myokine levels to depression/QoL in older athletes and controls. 55 athletes, 57 controls >59 years were enrolled. The assessment included ergometry, magnetic resonance imaging, blood withdrawal, and neuropsychological testing. Serum interleukin-6 (IL-6), irisin, brain-derived neurotrophic factor (BDNF), kynurenine, and cathepsin B were analyzed and compared to surrogates of depression and quality of life. Athletes presented with higher levels of Cathepsin B. Among controls, all myokines but irisin were associated with age. Also, among controls, kynurenine and IL-6 correlated inversely with specific dimensions of quality of life questionnaires, and IL-6 further with depressive symptoms and decreased physical performance. No such associations could be found among athletes. Irisin levels were inversely associated with mild depression and low-grade white matter-lesions in the brain and predicted impaired QoL. The circulating levels of several myokines/muscle activity-related factors appear to be associated with depressive symptoms and impaired QoL among older adults. However, in athletes, some of these connections seem ameliorated, suggesting additional stressors (as f.e. age) or a different pathomechanism among athletes.

Side-by-Side Comparison of Three Fully Automated SARS-CoV-2 Antibody Assays with a Focus on Specificity.

BACKGROUND: In the context of the COVID-19 pandemic, numerous new serological test systems for the detection of anti-SARS-CoV-2 antibodies rapidly have become available. However, the clinical performance of many of these is still insufficiently described. Therefore, we compared 3 commercial CE-marked, SARS-CoV-2 antibody assays side by side. METHODS: We included a total of 1154 specimens from pre-COVID-19 times and 65 samples from COVID-19 patients (≥14 days after symptom onset) to evaluate the test performance of SARS-CoV-2 serological assays by Abbott, Roche, and DiaSorin. RESULTS: All 3 assays presented with high specificities: 99.2% (98.6-99.7) for Abbott, 99.7% (99.2-100.0) for Roche, and 98.3% (97.3-98.9) for DiaSorin. In contrast to the manufacturers' specifications, sensitivities only ranged from 83.1% to 89.2%. Although the 3 methods were in good agreement (Cohen's Kappa 0.71-0.87), McNemar tests revealed significant differences between results obtained from Roche and DiaSorin. However, at low seroprevalences, the minor differences in specificity resulted in profound discrepancies of positive predictive values at 1% seroprevalence: 52.3% (36.2-67.9), 77.6% (52.8-91.5), and 32.6% (23.6-43.1) for Abbott, Roche, and DiaSorin, respectively. CONCLUSION: We found diagnostically relevant differences in specificities for the anti-SARS-CoV-2 antibody assays by Abbott, Roche, and DiaSorin that have a significant impact on the positive predictive values of these tests.

Type 2 diabetes care: Improvement by standardization at a diabetes rehabilitation clinic. An observational report.

BACKGROUND: Outcome of type 2 diabetes care depends on the acceptance of self-responsibility by informed patients, as treatment goals will otherwise be missed. AIMS AND METHODS: This pre/post-observational report describes the clinical outcome of type 2 diabetes care in patients with type 2 diabetes (N =930) admitted consecutively to a diabetes rehabilitation clinic (DRC) between June 2013, and June 2016, where they were exposed to standardized lifestyle modification with meals low in salt and rich in vegetables and fruits, totaling 1,200 to 1,600 kcal/d, and an add-on exercise load equivalent to 400-600 kcal/d. RESULTS: At admission, patients presented with multiple treatment modes, elevated HbA1c levels (7.6±1.5%, 60±16 mmol/mol), a high prevalence of co-morbidities dominated by obesity (79%), a low rate of influenza and pneumococcal immunization (<9%) and underuse of lipid-lowering drugs (-29%). Analysis of clinical and metabolic outcome after 3 weeks shows that simple standardization of and better adherence to treatment recommendations improved (p<0.0001) glucose (HbA1c -0.4±0.4%) and lipid metabolism (LDL/HDL ratio, -0.58±0.03), permitting a 39% reduction in insulin dosage, omission of insulin in 36/232 patients and omission of oral antidiabetic drugs (OADs) other than metformin and DPP4-inhibitors, while the use of GLP-1 analogs doubled to 5.2%. Improved outcome was independent of treatment strategy and more marked at initially high HbA1c at costs less than 25% of those encountered at a standard hospital. CONCLUSIONS: Our observations support the clinical notion that adherence to basic treatment recommendations is indispensable in type 2 diabetes care if metabolic and clinical treatment goals are to be met, and if inappropriate add-on over-medicalization with OADs and/or insulin is to be avoided. To this end, 'imprinting' patients at a DRC could be of considerable help.

Obesity: outcome of standardized life-style change in a rehabilitation clinic. An observational study.

Purpose: To explore differences in baseline characteristics following three weeks of semi-standardized in-patient care between patients with obesity without and with type 2 diabetes (T2D). Patients and methods: Patients without or with T2D were matched according to age, sex, and BMI. Food intake was restricted to 1,200-1,600 kcal/d to which a 400-600 kcal/d exercise load was added, and data were compared using Student's t-test, general linear models, and Spearman-rank correlations. Results: At baseline, patients with obesity and T2D displayed, besides elevated blood glucose and HbA1c values, higher serum liver enzymes (p<0.001-0.05), triglycerides, and CRP (p<0.01) and a greater prevalence of treated hyperlipidemia (p<0.001) than those with plain obesity who showed only higher LDL and HDL cholesterol levels (+9.0% and +16.0%). In response to three-weeks of standardized life-style change, both groups improved their vital variables and risk scores (p<0.001). While improvement in cholesterol slightly favored patients with plain obesity, the need for anti-hyperlipidemics (+25%) rose in both groups, albeit that for anti-hypertensives (+50%) increased only in patients with obesity and add-on T2D. Conclusion: Moderate changes in lifestyle improve the clinical condition, including coronary heart disease and premature mortality risk scores (HARD-CHD and ABSI) in patients with obesity both in the absence and presence of T2D, with the latter seemingly increasing the risk of hepatic steatosis and systemic inflammation.

Usage Data and Scientific Impact of the Prospectively Established Fluid Bioresources at the Hospital-Based MedUni Wien Biobank.

Background and Aim: It is increasingly recognized that biomedical research has serious reproducibility issues, which could be overcome at least in part by standardized processing of biomaterials. Therefore, professional biobanks have emerged, positively influencing sample and data quality. However, quantitative data about a biobank's contribution to published results are still hard to find, although they could serve as valuable benchmark figures for the community. We therefore aimed to report usage data from the MedUni Wien Biobank facility regarding its prospective fluid cohorts. Methods: Input and access statistics and publication output were reported for the years 2010-2017. Performance dynamics were tested by correlation analyses according to Spearman. Additionally, virtual costs per sample were calculated. Results: The amount of annually collected aliquots rose significantly from 68,500 in 2010 to 151,966 in 2017 (p = 0.015), although no further increase was recorded after 2012 (p = 0.266). In the same period, the quotient of requested to stored aliquots increased from 3.5% to 6.1% (p = 0.001), as the yearly number of requested aliquots nearly quadrupled from 2401 to 9342. Likewise, the number of published research articles per year to which the MedUni Wien Biobank contributed increased from 2 (total impact factor: 8.6) in 2010 to 16 (total impact factor: 69.0) in 2017, resulting in a total of 69 identified publications. Currently, the biobank operates at 15- to 20-fold overproduction, leading to virtual costs per accessed sample of ∼€20. Conclusion: The reported usage data might serve as a benchmark for other hospital-integrated biobanks, and implies that academic biobanks are able to produce considerable scientific impact at comparable moderate costs.

Type 1 diabetes care: Improvement by standardization in a diabetes rehabilitation clinic. An observational report.

BACKGROUND: T1D treatment requires informed self-responsible patients, who, however, frequently miss their therapeutic goals, providing considerable potential for improvement. METHODS: This observational report evaluates T1D patients [N = 109], aged ≥18 years (range 22-82), poorly controlled at home, at and 3 weeks after their admission to our diabetes rehabilitation clinic [DRC], where they were offered standardized, but unmonitored life-style modification. RESULTS: At admission, patients displayed elevated HbA1c values (66 mmol/mol [57; 81]), a high prevalence of co-morbidities (88%), lipodystrophies due to monolocal insulin injections (42%), a low rate of influenza (16%) and pneumococcal (7%) immunization, and underuse of lipid-lowering drugs (-38%). Standardization of life-style improved glucose (p<0.0001) and lipid metabolism (LDL/HDL ratio p<0.01) permitting reduction of insulin dose and reduction of add-on glucose-lowering drugs (GLDs) other than metformin. Outcome was independent of the mode of insulin treatment strategy and more marked at initially high HbA1c, with DRC-costs/d less than 25% of those encountered at standard hospitals. CONCLUSION: Type 1 diabetes care requires i) insulin treatment, food intake and life style to be handled in concert, ii) this need cannot be replaced by arbitrary addition of add-on GLDs, and iii) training to this end is 75% cheaper at a DRC than in standard hospitals.

One-Year stable perovskite solar cells by 2D/3D interface engineering.

Despite the impressive photovoltaic performances with power conversion efficiency beyond 22%, perovskite solar cells are poorly stable under operation, failing by far the market requirements. Various technological approaches have been proposed to overcome the instability problem, which, while delivering appreciable incremental improvements, are still far from a market-proof solution. Here we show one-year stable perovskite devices by engineering an ultra-stable 2D/3D (HOOC(CH2)4NH3)2PbI4/CH3NH3PbI3 perovskite junction. The 2D/3D forms an exceptional gradually-organized multi-dimensional interface that yields up to 12.9% efficiency in a carbon-based architecture, and 14.6% in standard mesoporous solar cells. To demonstrate the up-scale potential of our technology, we fabricate 10 × 10 cm2 solar modules by a fully printable industrial-scale process, delivering 11.2% efficiency stable for >10,000 h with zero loss in performances measured under controlled standard conditions. This innovative stable and low-cost architecture will enable the timely commercialization of perovskite solar cells.

A combination of routine blood analytes predicts fitness decrement in elderly endurance athletes.

Endurance sports are enjoying greater popularity, particularly among new target groups such as the elderly. Predictors of future physical capacities providing a basis for training adaptations are in high demand. We therefore aimed to estimate the future physical performance of elderly marathoners (runners/bicyclists) using a set of easily accessible standard laboratory parameters. To this end, 47 elderly marathon athletes underwent physical examinations including bicycle ergometry and a blood draw at baseline and after a three-year follow-up period. In order to compile a statistical model containing baseline laboratory results allowing prediction of follow-up ergometry performance, the cohort was subgrouped into a model training (n = 25) and a test sample (n = 22). The model containing significant predictors in univariate analysis (alanine aminotransferase, urea, folic acid, myeloperoxidase and total cholesterol) presented with high statistical significance and excellent goodness of fit (R2 = 0.789, ROC-AUC = 0.951±0.050) in the model training sample and was validated in the test sample (ROC-AUC = 0.786±0.098). Our results suggest that standard laboratory parameters could be particularly useful for predicting future physical capacity in elderly marathoners. It hence merits further research whether these conclusions can be translated to other disciplines or age groups.

Heterodimerization of AML1/ETO with CBFß is required for leukemogenesis but not for myeloproliferation.

The AML1/Runx1 transcription factor and its heterodimerization partner CBFß are essential regulators of myeloid differentiation. The chromosomal translocation t(8;21), fusing the DNA binding domain of AML1 to the corepressor eight-twenty-one (ETO), is frequently associated with acute myeloid leukemia and generates the AML1/ETO (AE) fusion protein. AE represses target genes usually activated by AML1 and also affects the endogenous repressive function of ETO at Notch target genes. In order to analyze the contribution of CBFß in AE-mediated leukemogenesis and deregulation of Notch target genes, we introduced two point mutations in a leukemia-initiating version of AE in mice, called AE9a, that disrupt the AML1/CBFß interaction (AE9aNT). We report that the AE9a/CBFß interaction is not required for the AE9a-mediated aberrant expression of AML1 target genes, while upregulation/derepression of Notch target genes does require the interaction with CBFß. Using retroviral transduction to express AE9a in murine adult bone marrow-derived hematopoietic progenitors, we observed that both AE9a and AE9aNT lead to increased myeloproliferation in vivo. However, both development of leukemia and long-term replating capacity are only observed with AE9a but not with AE9aNT. Thus, deregulation of both AML1 and Notch target genes is required for the development of AE9a-driven leukemia.

The effect of storage temperature fluctuations on the stability of biochemical analytes in blood serum.

BACKGROUND: Irreproducibility of scientific results constitutes an undesirably onerous economic burden and is in many cases caused by low-quality materials. Therefore, researchers are increasingly devoting their attention to the bioresources they use. In turn, those bioresources are required to validate their preanalytical processes in order to ensure best possible quality. The present study thus aimed to evaluate the impact of repeated temperature fluctuations, as they occur in most research biobanks due to repetitive opening and closing of freezer doors, on the stability of 26 biochemical analytes. METHODS: Serum of 43 individuals was randomly assigned to a fluctuation (n=21) and a control group (n=22). Serum of the fluctuation group underwent controlled temperature fluctuations (30 fluctuations <-75°C - <-65°C - <-75°C under real-life freezer conditions within 21 days). Control sera were stored at constant conditions. After 10, 20, and 30 fluctuations, results derived from the fluctuation group were compared to baseline and to the control group by means of general linear models. RESULTS: Sixteen biomarkers showed statistically significant changes over time, whereas only seven of those presented with diagnostically/clinically relevant changes at certain time points (aspartate aminotransferase, amylase, calcium, uric acid, creatinine, inorganic phosphate and total protein). However, there was no difference between the fluctuation and the control group. CONCLUSIONS: Some serum analytes are influenced by storage, even at temperatures as low as <-70°C. In contrast, we found no evidence that complex temperature fluctuations produced by storage of and access to biospecimens in biobank freezers generate any additional variability.

Deficiency of RITA results in multiple mitotic defects by affecting microtubule dynamics.

Deregulation of mitotic microtubule (MT) dynamics results in defective spindle assembly and chromosome missegregation, leading further to chromosome instability, a hallmark of tumor cells. RBP-J interacting and tubulin-associated protein (RITA) has been identified as a negative regulator of the Notch signaling pathway. Intriguingly, deregulated RITA is involved in primary hepatocellular carcinoma and other malignant entities. We were interested in the potential molecular mechanisms behind its involvement. We show here that RITA binds to tubulin and localizes to various mitotic MT structures. RITA coats MTs and affects their structures in vitro as well as in vivo. Tumor cell lines deficient of RITA display increased acetylated α-tubulin, enhanced MT stability and reduced MT dynamics, accompanied by multiple mitotic defects, including chromosome misalignment and segregation errors. Re-expression of wild-type RITA, but not RITA Δtub ineffectively binding to tubulin, restores the phenotypes, suggesting that the role of RITA in MT modulation is mediated via its interaction with tubulin. Mechanistically, RITA interacts with tubulin/histone deacetylase 6 (HDAC6) and its suppression decreases the binding of the deacetylase HDAC6 to tubulin/MTs. Furthermore, the mitotic defects and increased MT stability are also observed in RITA-/- mouse embryonic fibroblasts. RITA has thus a novel role in modulating MT dynamics and its deregulation results in erroneous chromosome segregation, one of the major reasons for chromosome instability in tumor cells.

Combined Effects of Inflammatory Status and Carotid Atherosclerosis: A 12-Year Follow-Up Study.

BACKGROUND AND PURPOSE: Inflammatory responses play a key role in atherogenesis. The aim of this study was to assess the prognostic value of hsCRP (high-sensitivity C-reactive protein) and to evaluate whether degree of carotid stenosis and serum levels of hsCRP jointly predict long-term mortality in asymptomatic patients with carotid atherosclerosis. METHODS: One thousand sixty-five patients with neurological asymptomatic carotid atherosclerosis as evaluated by duplex sonography were prospectively followed for cause-specific mortality. RESULTS: During a median of 11.81 years, a total of 549 deaths, including 362 cardiovascular deaths, were recorded. The risk of all-cause and cardiovascular mortality significantly increased in patients with elevated serum levels of hsCRP (the adjusted hazard ratio for cardiovascular mortality per increase of 1 mg/dL of hsCRP levels was 1.47; P<0.001). Patients with a high degree of carotid stenosis and increased hsCRP levels were particularly at risk of adverse outcome. Patients with carotid narrowing over 50% and hsCRP levels >0.29 mg/dL (=median) had nearly twice as high a risk of cardiovascular mortality compared with patients with carotid stenosis of <50% and hsCRP levels <0.29 mg/dL (adjusted hazard ratio 1.89; P<0.001). Improvement in risk stratification with combined assessment of carotid stenosis and hsCRP was confirmed by an improvement of the continuous net reclassification improvement with 18% for all-cause mortality and 15% for cardiovascular mortality compared with the degree of carotid stenosis alone (P<0.01). CONCLUSIONS: Measurement of hsCRP in combination with ultrasound investigations of the carotid arteries at a single time point provides additional prognostic information for patients with asymptomatic carotid atherosclerosis.