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Background: The A/H1N1/2009 influenza pandemic made evident the need for faster and higher-yield methods for the production of influenza vaccines. Platforms based on virus culture in mammalian or insect cells are currently under investigation. Alternatively, expression of fragments of the hemagglutinin (HA) protein in prokaryotic systems can potentially be the most efficacious strategy for the manufacture of large quantities of influenza vaccine in a short period of time. Despite experimental evidence on the immunogenic potential of HA protein constructs expressed in bacteria, it is still generally accepted that glycosylation should be a requirement for vaccine efficacy. Methodology/Principal Findings: We expressed the globular HA receptor binding domain, referred to here as HA63-286- RBD, of the influenza A/H1N1/2009 virus in Escherichia coli using a simple, robust and scalable process. The recombinant protein was refolded and purified from the insoluble fraction of the cellular lysate as a single species. Recombinant HA63-286-RBD appears to be properly folded, as shown by analytical ultracentrifugation and bio-recognition assays. It binds specifically to serum antibodies from influenza A/H1N1/2009 patients and was found to be immunogenic, to be capable of triggering the production of neutralizing antibodies, and to have protective activity in the ferret model. Conclusions/Significance: Projections based on our production/purification data indicate that this strategy could yield up to half a billion doses of vaccine per month in a medium-scale pharmaceutical production facility equipped for bacterial culture. Also, our findings demonstrate that glycosylation is not a mandatory requirement for influenza vaccine efficacy. [ABSTRACT FROM AUTHOR]

Background: Guideline recommendations for the management of lipids in patients at risk for cardiovascular disease is largely based on low-density lipoprotein cholesterol (LDL-C) concentration. LDL-C is commonly calculated by the Friedewald equation, which has many limitations. The National Institutes of Health (NIH) equation better estimates LDL-C, particularly in patients with hypertriglyceridemia and/or low LDL-C. We validated the NIH LDL-C equation at the first Canadian clinical laboratory to implement this equation., Methods: A total of 3161 lipid ultracentrifugation results from a specialized lipid cohort of 2836 patients were included. LDL-C was calculated using the NIH and Friedewald equations and compared to LDL-C measured by ultracentrifugation. We determined the accuracy of these equations at treatment thresholds and developed NIH equation restriction criteria to ensure only accurate results are reported., Results: Ultracentrifugation LDL-C more strongly correlated with NIH-calculated LDL-C (r 2  = 0.889) than Friedewald-calculated LDL-C (r 2  = 0.807) and resulted in fewer non-sensical negative LDL-C values. The correlation for NIH-calculated LDL-C improved to r 2  = 0.975 after applying our restriction criteria. The NIH equation showed equivalent or superior concordance with ultracentrifugation at treatment thresholds. The LDL-C mean absolute difference increased with increasing TG and decreasing LDL-C concentrations, although the NIH equation was more robust under both conditions., Conclusions: We validated the NIH equation against ultracentrifugation in a cohort with a wide lipid concentration range, which supported its superiority over the Friedewald equation. We recommend clinical implementing the NIH equation for all patients except those with type III hyperlipoproteinemia or TG > 9.04 mmol/L, with an LDL-C lower reporting limit of <0.50 mmol/L., (Copyright © 2021 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.)

Background: High levels of the triglycerides to high-density lipoprotein cholesterol (TG/HDL-C) ratio are associated with obesity, metabolic syndrome, and insulin resistance., Objectives: We evaluated variability in the remaining lipid profile, especially remnant lipoprotein particle cholesterol (RLP-C) and its components (very low-density lipoprotein cholesterol subfraction 3 and intermediate-density lipoprotein cholesterol), with variability in the TG/HDL-C ratio in a very large study cohort representative of the general U.S., Methods: We examined data from 1,350,908 US individuals who were clinically referred for lipoprotein cholesterol ultracentrifugation (Atherotech, Birmingham, AL) from 2009 to 2011. Demographic information other than age and sex was not available. Changes to the remaining lipid profile across percentiles of the TG/HDL-C ratio were quantified, as well as by three TG/HDL-C cut-off points previously proposed in the literature: 2.5 (male) and 2 (female), 3.75 (male) and 3 (female), and 3.5 (male and female)., Results: The mean age of our study population was 58.7 years, and 48% were men. The median TG/HDL-C ratio was 2.2. Across increasing TG/HDL-C ratios, we found steadily increasing levels of RLP-C, non-HDL-C and LDL density. Among the lipid parameters studied, RLP-C and LDL density had the highest relative increase when comparing individuals with elevated TG/HDL-C levels to those with lower TG/HDL-C levels using established cut-off points. Approximately 47% of TG/HDL-C ratio variance was attributable to RLP-C., Conclusions: In the present analysis, a higher TG/HDL-C ratio was associated with an increasingly atherogenic lipid phenotype, characterized by higher RLP-C along with higher non-HDL-C and LDL density., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

Background: Accurate high-density lipoprotein cholesterol (HDL-C) measurements are important for management of cardiovascular diseases. The US Centers for Disease Control and Prevention (CDC) and Cholesterol Reference Method Laboratory Network (CRMLN) perform ultracentrifugation (UC) reference measurement procedure (RMP) to value assign HDL-C. Japanese CRMLN laboratory (Osaka) concurrently runs UC procedure and the designated comparison method (DCM). Osaka performance of UC and DCM was examined and compared with CDC RMP., Methods: CDC RMP involved UC, heparin-MnCl₂ precipitation, and cholesterol analysis. CRMLN DCM for samples containing <200 mg/dl triglycerides involved 50-kDa dextran sulfate-MgCl2 precipitation and cholesterol determination., Results: HDL-C regression equations obtained with CDC (x) and Osaka (y) were y=0.992x+0.542 (R(2)=0.996) for Osaka UC and y=1.004x-0.181 (R(2)=0.998) for DCM. Pass rates within ±1 mg/dl of the CDC target value were 91.9 and 92.1% for Osaka UC and DCM, respectively. Biases at 40 mg/dl HDL-C were +0.22 and -0.02 mg/dl for Osaka UC and DCM, respectively., Conclusions: Osaka UC and DCM were highly accurate, precise, and stable for many years, assisting manufacturers to calibrate products for clinical laboratories to accurately measure HDL-C for patients, calculate non-HDL-C, and estimate low-density lipoprotein cholesterol with the Friedewald equation., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Background: The FDA requirement for sensitivity of viral NAT methods used in blood screening is a 95-percent detection limit of 100 copies per mL, whereas the NAT screening system should have a sensitivity of at least 5000 copies per mL per individual donation. According to the Common Technical Specifications of the European Directive 98/79/EC for in vitro diagnostics, viral standard dilutions (calibrated against the WHO standard) should be tested at least 24 times for a statistically valid assessment of the 95-percent detection limit., Study Design and Methods: Viral standard dilution panels (PeliCheck, VQC-CLB) were prepared for HCV RNA genotypes 1 and 3 and for HIV RNA genotypes B and E. In a multicenter study, 23 laboratories tested the panels all together in 8 to 91 test runs per NAT method., Results: The following 95-percent detection limits (and 95% CIs) were found on the HCV RNA genotype 1 reference panels (shown as geq/mL): Gen-Probe TMA, 85 (64-118); AmpliScreen, 126 (83-225); AmpliScreen with NucliSens Extractor, 21 (13-44); Amplicor with NucliSens Extractor, 69 (50-102), and Amplicor with Qiagen extraction technology, 144 (74-102). On HIV RNA genotype B dilution panels, the following 95-percent detection limits were found (shown as geq/mL): Gen-Probe TMA, 31 (20-52); AmpliScreen, 126 (67-311); AmpliScreen with NucliSens Extractor, 37 (23-69), and NucliSens QL assay, 123 (51-566). HIV RNA genotype E panels were detected with equal sensitivity as HIV RNA genotype B panels. In the Gen-Probe TMA assay, the 50-percent detection limits on HIV RNA type B and type E were 3.6 (2.6-5.0) and 3.9 (2.4-5.8) geq per mL, respectively. The HCV RNA genotype 1 and 3 standards were detected with equal sensitivity., Conclusion: The differences in sensitivity between NAT assays can be explained by the input of isolated viral nucleic acid in the amplification reactions. The FDA requirements for sensitivity of NAT blood screening assays can be met by the Gen-probe TMA, as well as by the AmpliScreen assays, particularly when combined with the NucliSens Extractor.

Low-density lipoprotein (LDL) cholesterol concentrations are most commonly estimated by the formula LDL cholesterol = total cholesterol - [triglycerides (TG)/5 + high-density lipoprotein cholesterol], although alternative factors such as TG/6 have also been used. Using standardized, automated, enzymatic lipid assays, we analyzed 4797 plasma samples from normal and dyslipidemic adults, to compare LDL cholesterol concentrations obtained after ultracentrifugation with those calculated by several such methods (i.e., TG/4-TG/8). or TG concentrations less than or equal to 0.50 g/L, TG/4 agreed best with the direct assay; for TG of 0.51-2.00 g/L, TG/4.5 was best; and for TG of 2.01-4.00 g/L, TG/5 was best. Differences in estimated values were generally small, however. At TG greater than 4.00 g/L, none of the factors tested allowed a reliable estimate of LDL cholesterol. When TG were less than or equal to 4.00 g/L, 86% of estimated LDL cholesterol values were properly classified according to National Cholesterol Education Program cutpoints when the factor TG/5 was used. We conclude that a convenient direct method for measuring LDL cholesterol is needed but, until one is available, use of the factor TG/5 will assure that most individuals with TG less than or equal to 4.00 g/L, as measured in a standardized laboratory, can be reasonably well classified for risk of coronary artery disease.

Cross hemagglutination inhibition (HI) and neuraminidase inhibition (NI) tests demonstrated strong antigenic relationship between A/Swine/Wisconsin/1/73 (SW/73) and A/Swine/Shope/15/31 (SW/31) influenza viruses. An eigthyone fold purification of virus was achieved by adsorption and elution followed by differential ultracentrifugation and sedimentation through linear sucrose gradient. Radioimmunoassay using purified 125I labeled viral antigens revealed antigenic variation between the two virus isolates. Neuraminidase of both viruses had pH optima between 6.5 and 7.0, and SW/31 enzyme was relatively more heat stable than SW/73.