Evaluation of Roche Hemolysis Index for Reporting of Plasma Hemoglobin, and Considerations Regarding Hemolysis Detection in Use of Ventricular-Assist Devices

Introduction/Objective Our cardiac intensive care unit (CICU) asked the laboratory to provide plasma hemoglobin testing to monitor effects of ventricular-assist devices (VAD). Roche hemolysis index (HI) has been reported to be suitable for this purpose. Use of HI as a reportable test, however, must be validated in-house as a laboratory- developed test (LDT), according to CLIA regulations. We describe results of our evaluation, and caveats for intended use. Methods/Case Report Concentrated hemolysate was produced by osmotic lysis of packed red blood cells in water. Hemolysis in plasma was by dilution of concentrated hemolysate. Verification of HI as a measure of hemoglobin (mg/dL) used the Sigma-Aldrich spectrophotometric hemoglobin assay as a gold standard. Linearity, linear range, sensitivity, were investigated by dilution measurements; interferences by admixture experiments; reproducibility (precision, intra- and inter-assay) in each case by replicate measurements (n = 20). Results (if a Case Study enter NA) HI was confirmed to correspond to hemoglobin in mg/dL. Linearity was between 2-1000 mg/dL (r2 >0.99). Intra-assay precisions were <=2.5% (hemoglobin = 74,148 mg/dL); inter-assay precisions were <=4.9% (hemoglobin = 69,139 mg/dL). HI variability was ±2 at very low values (0-10 mg/dL). There were no interferences observed among common therapeutic drugs. Hyperlipemia (evaluated up to lipemic index (LI) = 225) and hyperbilirubinemia (evaluated up to icteric index (II) = 7) demonstrated no significant interference. Conclusion Analytically, HI exhibited acceptable performance for reporting of plasma hemoglobin. Deployment would require establishment of quality control (QC) and proficiency testing procedures for HI. Clinically, a caveat for use is that HI cannot distinguish between hemolysis in circulation vs. that due to sample collection. For the CICU, HI >10 mg/dL is characteristic of more than 20% of samples drawn in general, irrespective of use of VAD. Temporal patterns of HI must therefore be carefully evaluated in parallel with haptoglobin measurements to assist in VAD management.

An inter-day assessment of the ABC parameters in the evaluation of progressive keratoconus

The progression of keratoconus is commonly determined by comparing the results of corneal tomographic measurements on different occasions. However, investigations on the repeatability of measurements are commonly performed within the same day, thus not taking the inter-day variation into account. The effect of keratoconus disease severity on the measurement error is also seldom considered. In this post hoc investigation, the parameters A, B and C in the Belin ABCD Progression Display were evaluated in relation to disease severity in intra-day and inter-day measurements. Four consecutive measurements were performed on 61 patients with keratoconus on the same day (intra-day). In another cohort, four consecutive measurements were obtained and then repeated 3 days later in 25 patients with keratoconus and 25 healthy controls (inter-day). The results suggest that the diagnosis of disease progression would benefit from inter-day measurements, and the stratification of the parameters A and C according to disease severity. It is also recommended that tomographic systems such as the Pentacam HR be modified to allow the comparison of both single measurements and the mean of replicate measurements of the parameters used in the assessment of progression of keratoconus.

Comparing singlet and duplicate immunogenicity assay in human plasma for pembrolizumab using Gyrolab®

Aim: For decades, the traditional approach for ligand-binding assays has been to generate two measurements from adjacent wells on the plate. In recent years, scientists have investigated the true benefit of this ‘duplicate analysis’ by looking back at previously generated bioanalytical data with the conclusion that the benefits are negligible. Materials & methods: We demonstrated a method development approach to determine the best number of replicate measurements of an immunogenicity assay. We used an anti-pembrolizumab immunogenicity assay on Gyrolab® to challenge the traditional use of duplicate measurements as we compare it to singlet measurement and show a balanced design for assessing the cut-point in singlet. Results & conclusion: We introduced the concept of calculating the maximum drug tolerance during method development. In this method, we found no practical benefit for duplicate analysis and go further in recommending that singlet analysis should be considered the default for all ligand-binding assays.

Towards fast, routine blood sample quality evaluation by Probe Electrospray Ionization (PESI) metabolomics

PESI-MS enables with its greatly simplified handling and fast result delivery the application field for high-throughput use in routine settings. In health care and research, pre-analytical errors often remain undetected and disrupt diagnosis, treatment, clinical studies and biomarker validations incurring high costs. This proof-of-principle study investigates the suitability of PESI-MS for robust, routine sample quality evaluation.One of the most common pre-analytical quality issues in blood sampling are prolonged transportations times from bedside to laboratory promptly changing the metabolome. Here, human blood (n=50) was processed immediately or with a time delay of 3 h. The developed sample preparation method delivers ready-to-measure extracts in <8 min. PESI-MS spectra were measured in both ionization modes in 2 min from as little as 2 µl plasma allowing 3 replicate measurements. The mass spectra contained 1200 stable features covering a broad chemical space covering major metabolic classes (e.g. fatty acids, lysolipids, lipids). The time delay of 3 h was predictable by using 18 features with AUC > 0.95 with various machine learning and was robust against loss of single features.Our results serve as first proof of principle for the unique advantages of PESI-MS in sample quality assessments. The results pave the way towards a fully automated, cost-efficient, user-friendly, robust and fast quality assessment of human blood samples from minimal sample amounts.Graphical abstract

Continuous-Flow Analysis of δ17O, δ18O, and δD of H2O on an Ice Core from the South Pole

The δD and δ18O values of water are key measurements in polar ice-core research, owing to their strong and well-understood relationship with local temperature. Deuterium excess, d, the deviation from the average linear relationship between δD and δ18O, is also commonly used to provide information about the oceanic moisture sources where polar precipitation originates. Measurements of δ17O and “17O excess” (Δ17O) are also of interest because of their potential to provide information complementary to d. Such measurements are challenging because of the greater precision required, particularly for Δ17O. Here, high-precision measurements are reported for δ17O, δ18O, and δD on a new ice core from the South Pole, using a continuous-flow measurement system coupled to two cavity ring-down laser spectroscopy instruments. Replicate measurements show that at 0.5 cm resolution, external precision is ∼0.2‰ for δ17O and δ18O, and ∼1‰ for δD. For Δ17O, achieving external precision of &lt;0.01‰ requires depth averages of ∼50 cm. The resulting ∼54,000-year record of the complete oxygen and hydrogen isotope ratios from the South Pole ice core is discussed. The time series of Δ17O variations from the South Pole shows significant millennial-scale variability, and is correlated with the logarithmic formulation of deuterium excess (dln), but not the traditional linear formulation (d).

Development and validation of a GC–MS method for the simultaneous determination of acetochlor, fluorochloridone, and pendimethalin in a herbicide emulsifiable concentrate formulation

This paper describes a rapid method to simultaneously determine acetochlor, fluorochloridone and pendimethalin present in a herbicide emulsifiable concentrate (EC) formulation using gas chromatography–mass spectrometry (GC–MS). Selected ion monitoring mode was performed to increase the sensitivity, with dibutyl phthalate as an internal standard. The method was validated with respect to linearity, accuracy, precision, and stability. Chromatographic separation was carried out on a TG-5 MS column (30 m × 0.25 mm × 0.25 μm) with helium as the carrier gas at a flow rate of 1.0 mL/min. Calibration curves were linear over 2.0–20.0 μg/mL for each analyte, and the limit of quantification was below 20 ng/mL. Good performance in terms of recovery ranging from 94.5% to 102.5% at 3 concentration levels proved excellent accuracy. The intra- and inter-day relative standard deviations for 6 replicate measurements were always less than 5%. The developed method is simple and efficient for the routine determination of the ternary mixtures in a compound herbicide EC formulation product.

Determination of the Development Performances of Pecan Saplings in Aydin ecological Conditions

In this research, the sapling growth performances of Pecan cultivation in Aydın ecological conditions were investigated. "Burkett" Pecan rootstock and "Mahan14", "Wichita7" and "Choctaw2" pecan cultivars were used. Starting from the date of planting; Sapling stem diameters were measured 5 cm above the graft site. Diameters of rootstocks was measured 5 cm below the graft site, bud burst dates are determined (min %70 of buds), shoot diameters were measured at 15-day intervals from the place after the first two buds. Shoot lengths were measured at 15-day intervals. The measurements were carried out for 2 consecutive years. Repeared Measures Anova is used to determine whether there are significant differences in the data. The development difference between cultivars in both regions was investigated. In the analysis, averages of five replicate measurements in two years and 12 different periods. Pecan saplings showed a good development in the ecological conditions of Aydin (37°45′ 0″N - 28°0′0″E). These results show that in the province of Aydın, Pecan cultivation can be done except in very cold and very dry places. However, the study should be supported by future fruit productivity studies. The present research findings will be useful for regions with similar climatic conditions.

Application of two charge transfer complex formation reactions for selective determination of metformin hydrochloride in pharmaceuticals and urine

Background Metformin hydrochloride (MFH) is a biguanide class anti-diabetic drug used to treat type-2 diabetes mellitus. Its reaction with two charge-transfer complexing agents, p-chloranilic acid (PCA) and 2,3-dichloro-5,6-dicyano-p-benzoquinone (DDQ) in acetonitrile medium to yield coloured products measurable at wavelengths of maxima 530.0 and 460.0 nm, respectively, was conveniently used to develop two spectrophotometric methods for analyses of bulk sample and tablets. Results The effect of solvent, reagent concentration and reaction time to form charge-transfer (CT) complexes was meticulously studied and optimized. Under optimised conditions, the absorbance at the respective wavelength of maximum versus concentration of MFH was in linear correlation for the range from 8.0 to 320.0 and from 1.6 to 64.0 μg mL-1 in PCA and DDQ methods, respectively, and correspondingly, the values of molar absorptivity of 0.733 × 103 and 0.257 × 104 L mol-1 cm-1 and Sandell sensitivity of 0.3620 and 0.0644 μg cm-2. The quantification (QL) and detection (DL) limits were 2.67 and 0.88 μg mL-1 for PCA method, and 0.33 and 0.11 μg mL-1 for DDQ method. Conclusion The new methods were emerged as repeatable and reproducible, with replicate measurements for intra- and inter-day variations as showed by obtained RSD values of < 2%. Within a day and between day relative errors were ≤ 2.18%. Methods were also validated for robustness, ruggedness and selectivity and agreeing results were produced. The methods were used to analyse MFH-containing tablets very accurately and precisely as reflected by the mean recovery value close to 100% and lower RSD values, respectively. Analysis of spiked human urine yielded excellent mean recoveries, indicating the absence of interference from endogenous substances.

Handling deviating control values in concentration-response curves

In cell biology, pharmacology and toxicology dose-response and concentration-response curves are frequently fitted to data with statistical methods. Such fits are used to derive quantitative measures (e.g. EC$$_{20}$$ 20 values) describing the relationship between the concentration of a compound or the strength of an intervention applied to cells and its effect on viability or function of these cells. Often, a reference, called negative control (or solvent control), is used to normalize the data. The negative control data sometimes deviate from the values measured for low (ineffective) test compound concentrations. In such cases, normalization of the data with respect to control values leads to biased estimates of the parameters of the concentration-response curve. Low quality estimates of effective concentrations can be the consequence. In a literature study, we found that this problem occurs in a large percentage of toxicological publications. We propose different strategies to tackle the problem, including complete omission of the controls. Data from a controlled simulation study indicate the best-suited problem solution for different data structure scenarios. This was further exemplified by a real concentration-response study. We provide the following recommendations how to handle deviating controls: (1) The log-logistic 4pLL model is a good default option. (2) When there are at least two concentrations in the no-effect range, low variances of the replicate measurements, and deviating controls, control values should be omitted before fitting the model. (3) When data are missing in the no-effect range, the Brain-Cousens model sometimes leads to better results than the default model.

EmpiReS: Differential Analysis of Gene Expression and Alternative Splicing

ABSTRACTWhile absolute quantification is challenging in high-throughput measurements, changes of features between conditions can often be determined with high precision. Therefore, analysis of fold changes is the standard method, but often, a doubly differential analysis of changes of changes is required. Differential alternative splicing is an example of a doubly differential analysis, i.e. fold changes between conditions for different isoforms of a gene. EmpiRe is a quantitative approach for various kinds of omics data based on fold changes for appropriate features of biological objects. Empirical error distributions for these fold changes are estimated from Replicate measurements and used to quantify feature fold changes and their directions. We assess the performance of EmpiRe to detect differentially expressed genes applied to RNA-Seq using simulated data. It achieved higher precision than established tools at nearly the same recall level. Furthermore, we assess the detection of alternatively Spliced genes via changes of isoform fold changes (EmpiReS) on distribution-free simulations and experimentally validated splicing events. EmpiReS achieves the best precision-recall values for simulations based on different biological datasets. We propose EmpiRe(S) as a general, quantitative and fast approach with high reliability and an excellent trade-off between sensitivity and precision in (doubly) differential analyses.