Stability of Wheat Floret Metabolites during Untargeted Metabolomics Studies

A typical metabolomic analysis consists of a multi-step procedure. Variation can be introduced in any analysis segment if proper care in quality assurance is not taken, thus compromising the final results. Sample stability is one of those factors. Although sophisticated studies addressing sample decay over time have been performed in the medical field, they are emerging in plant metabolomics. Here, we focus on the stability of wheat floret extracts on queue inside an auto-injector held at 25 °C. The objective was to locate an analytical time window from extraction to injection with no significant difference occurring in the sample. Total ion current chromatograms, principal component analysis, and volcano plots were used to measure changes in the samples. Results indicate a maximum work window time of 7:45 h for Steele-ND wheat methanolic extractions in an auto-sampler at 25 °C. Comparisons showed a significant gradual increase in the number and intensity of compounds observed that may be caused by the degradation of other molecules in the sample extract. The approach can be applied as preliminary work in a metabolite profiling study, helping to set the appropriate workload to produce confident results.

Duration of respiratory sample stability at -80ºC for SARS-CoV-2 PCR

Objective: To determine the stability of respiratory samples for SARS-CoV-2 PCR at standard laboratory ultra-freezer temperatures (-80°C). Methods: Five hundred and sixty-five archived, SARS-CoV-2 PCR positive patient specimens received at the Pathology Department of the Indus Hospital & Health Network between January 2021 and June 2021 were retested in June 2021. Samples had been stored at -70°C or below throughout this duration. Sample integrity following storage was assessed as the percentage of samples with reproducible results, and as consistency of cycle threshold (Ct) values between the original testing and the repeat testing. Results: Of the 565 samples evaluated in this study, 86% gave reproducible results upon retesting. However, there was no correlation between the duration of storage and result reproducibility, though the majority (69% for PCR Target-I and 78% for PCR Target-II respectively) of non-reproducible results had Ct values above 30. Similarly, there was a consistent increase of Ct values upon storage at ultra-freezer temperatures, though the effect again was more contingent upon freezing the sample in the ultra-freezer rather than the duration of storage. Conclusion: SARS-CoV-2 positive respiratory specimens for PCR can be stored for up to six months at -70°C or below without loss of sample integrity, though there is some loss of PCR-detected viral targets as evidenced by an immediate increased in the PCR-generated Ct values. In addition, samples with initial Ct values above 30 are more likely to give non-reproducible results. doi: https://doi.org/10.12669/pjms.38.ICON-2022.5777 How to cite this:Aijaz J, Naseer F, Dojki M, Jamal S. Duration of respiratory sample stability at -80ºC for SARS-CoV-2 PCR. Pak J Med Sci. 2022;38(2):393-398. doi: https://doi.org/10.12669/pjms.38.ICON-2022.5777 This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Adalimumab and anti-adalimumab LISA-TRACKER immunoassays performance criteria for therapeutic drug monitoring of adalimumab-amgen biosimilar (ABP501)

Background ABP501 is a biosimilar to Reference Adalimumab (HUMIRA®) produced by AMGEN. Adalimumab (ADA) has a marketing authorization for Crohn's disease, ulcerative colitis and other inflammatory or autoimmune diseases. The aim of this study was to evaluate the LISA-TRACKER assays developed by Theradiag (France), for the monitoring of ABP501 and anti-ABP501 antibodies in human serum. Results 68 ABP501 clinical samples were measured with the LISA TRACKER Duo Adalimumab assay. LISA TRACKER has been validated as suitable for quantification of ABP501 in human serum samples. Accuracy of the LISA-TRACKER was measured using 3 human serum matrices spiked with known levels of biosimilar, 3 levels spanning the dynamic range. Percentages of recovery were ranged from 90 to 120% for biosimilar batch1, and between 93 and 105% for biosimilar batch2. The acceptance criteria (CV < 20%) were met for intra-run (from 3.8 to 16.5%) and inter-run imprecision (from 4.4 to 13.9%) including the two batches. All results were comprised within ± 20% from results, obtained with the kit and sample unexposed in order to evaluate stability of the sample, stability of the kit and consistency of the results. In any case, but two, all percentages of inhibition were > 50% for specificity. Specificity was tested with Biosimilar spiked samples, Biosimilar with Humira® spiked samples, and clinical samples from patients treated with adalimumab biosimilar. All of these samples were spiked with polyclonal antibodies directed against Humira®. Specificity inhibition and specificity detection steps were also part of the validation parameters. Reagents made with ABP501 gave similar results than reagents made with Humira® meeting acceptance criteria. Conclusions LISA-TRACKER ADA and anti-ADA assays are reliable for the monitoring of patients treated with ABP501.

Total lab automation: sample stability of clinical chemistry parameters in an automated storage and retrieval module

Objectives Automated storage and retrieval modules (SRM), as part of total lab automation (TLA) systems, offer tremendous practical and economic benefits. In contrast to manual storage systems, SRMs indicate continuous motion of samples and may leave samples prone to temperature fluctuations. This study investigates analyte stability in serum and heparin plasma within an automated storage module. Methods The stability of 28 common biochemistry analytes was investigated using 57 freshly obtained routine serum samples and 42 lithium-heparin plasma samples. Following baseline measurement, samples were stored at 2–8 °C in the automated SRM of the Accelerator a3600 TLA and reanalyzed at fixed time points (2, 4, 8, 12, 24, 48 and 72 h) on the Abbott Architect c16000 chemistry analyzer. The concentration at each time point was expressed as %-difference to the baseline value and mean results were compared to the criteria for desirable bias derived from the biological variation database. Results Nine of the analytes exceeded the bias criterion within 72 h after initial measurement in either serum samples, plasma samples or both. Lithium-heparin plasma samples showed increasing values for phosphor, potassium and lactate dehydrogenase (LDH), which were only considered stable for respectively 24, 12 and 4 h, glucose was considered stable for 8 h. Electrolyte concentrations and LDH activity significantly increased in serum samples beyond 48 h. Bicarbonate should not be performed as add-on test at all. Conclusions The presented data indicate that the conditions within an SRM have no clinical impact on sample stability and allow stable measurement of routine analytes within 72 h, comparable to manual storage facilities.

Flow cytometry and pharmacokinetics

Multiparametric flow cytometry is a powerful cellular analysis tool used in various stages of drug development. In adoptive cell therapies, the flow cytometry methods are used for the evaluation of advanced cellular products during manufacturing and to monitor cellular kinetics after infusion. In this report, we discussed the bioanalytical method development challenges to monitor cellular kinetics in CAR-T cell therapies. These method development challenges include procuring positive control samples for the development of the method, flow cytometry panel design, LLOQ, prestain sample stability, staining reagents and data analysis.

Features of «Plants» cluster from the reference materials collection IGC SB RAS

Four multielement reference materials compile the «Plants» cluster in the developed by IGC SB RAS collection. These are part of terrestrial plants (birch leaf, pine needles, mown meadow grass) and the aquatic plant Elodea canadensis (roots, stems, leaves and flowers). Plants that are sensitive indicators of the state of the environment are collected from unpolluted territories of Eastern Siberia (near and on Lake Baikal). The paper discusses differences in methods of selection and preparation of the material. Such features of these reference materials as granulometric composition (shape, size, particle size distribution), homogeneity and minimum representative mass of the sample, stability ofpowders under conditions of natural aging were studied in accordance with Russian and international requirements with the use of modern devices and methods of chemical analysis. The elemental compositions of matrix plant samples were evaluated according to the method of interlaboratory certification and are represented by the contents of more than 60 elements, of which 23 to 41 are certified. The participation of 20 to 38 accredited Russian and foreign laboratories and the use of more than ten different methods of analysis ensured the traceability of the results. The comparison of the developed and Chinese certified matrix plant reference samples demonstrated their consistency. Based on the results of the discussion of the characteristic properties offour plant PM, they are recommended for performing chemical measurements during the validation of existing and development of new analytical methods, quality control and evaluation of the traceability of the results of determining a wide range of elements in plant materials, as well as professional testing of laboratories of geo-ecological, pharmaceutical and agricultural organisations.

Study of the parameters of absorption into the bloodstream and excretion of the drug Gamavit after a single administration to laboratory mini-pigs

A pharmacokinetic study of the absorption into the bloodstream, bioavailability and excretion of Gamavit from the body after intramuscular administration to laboratory mini-pigs was conducted. Quantitative determination was carried out by HPLC using a fluorimetric detector, for which Gamavit was labeled with Cy5 dye, which was then used for mini-pigs inoculation. The developed methods for determining Gamavit in the blood and feces were validated according to the following validation parameters: selectivity, calibration curve, accuracy, precision, limit of quantitative determination, sample transfer, and sample stability. The confirmed analytical range of the method for Gamavit detection in blood plasma and feces was 1.00…50.0 mcg/ml. Maximum concentration of Gamavit in the blood of mini-pigs after a single intramuscular injection was 30.97 mcg/ml and was reached on average 15 minutes after administration. 24 hours following administration, Gamavit was still detected in the blood in insignificant amounts. The average half-life of Gamavit in the blood is 8.64±3.50 hours. After administration at a dose of 0.1 ml/kg, the clearance of the drug is 1.27 l/kg * h, the excretion rate at an effective concentration of 30 mg/l is 38 mg/kg*h, and the maintenance dose when using the drug 1 time a day is 0.9…1.0 ml. The detection of the label in the feces of the studied animals indicates that one of the ways Gamavit removal is excretion with the help of bile acids, as well as partial excretion with feces.

Sample stability for congenital cytomegalovirus assay using Alethia after prolonged storage at different temperatures

Congenital cytomegalovirus (cCMV) infection is a leading non-genetic cause of sensorineural hearing. Alethia CMV assay is proved to be accurate, simple and appears suitable for CMV testing using neonatal saliva. However, long storage of swab samples is not validated across different time periods and storage temperature. This study verifies the effects of storage time and temperature in detection of CMV from saliva samples using molecular assay developed by Alethia.