Plasma free DNA: Evaluation of temperature-associated storage effects observed for Roche Cell-Free DNA collection tubes

Introduction: Standardized pre-analytical blood sample procedures for the analysis of circulating cell-free DNA (ccfDNA) are still not available. Therefore, the present study aimed at evaluating the impact of storage conditions related to different times (24 and 48 h) and temperatures (room temperature (RT) and 4 - 8 °C) on the plasma ccfDNA concentration of blood samples drawn into Cell-Free DNA collection tubes (Roche Diagnostics GmbH, Mannheim, Germany). Materials and methods: Venous blood from 30 healthy individuals was collected into five 8.5 mL Cell-Free DNA Collection Tubes (Roche Diagnostics GmbH) each. Plasma samples were processed at time point of blood collection (tube 1), and after storage under the following conditions: 24 h at RT (tube 2) or 4-8 °C (tube 3), and 48 h at RT (tube 4) or 4 - 8 °C (tube 5). Circulating cell-free DNA concentrations were determined by EvaGreen chemistry-based droplet digital PCR (ddPCR). Results: No statistically significant differences between median (interquartile range) plasma ccfDNA concentrations (ng/mL) at time point of blood collection (3.17 (2.13 – 3.76)) and after storage for 24 h (RT: 3.02 (2.41 – 3.68); 4-8 °C: 3.21 (2.19 – 3.46)) and 48 h (RT: 3.13 (2.10 – 3.76); 4-8 °C: 3.09 (2.19 – 3.50)) were observed (P values from 0.102 – 0.975). Conclusions: No unwanted release of genomic DNA from white blood cells could be detected in plasma samples after tube storage for 24 and 48 h regardless of storage temperature

Biochip-Based Detection of KRAS Mutation in Non-Small Cell Lung Cancer

This study is aimed at evaluating the potential of a biochip assay to sensitively detect KRAS mutation in DNA from non-small cell lung cancer (NSCLC) tissue samples. The assay covers 10 mutations in codons 12 and 13 of the KRAS gene, and is based on mutant-enriched PCR followed by reverse-hybridization of biotinylated amplification products to an array of sequence-specific probes immobilized on the tip of a rectangular plastic stick (biochip). Biochip hybridization identified 17 (21%) samples to carry a KRAS mutation of which 16 (33%) were adenocarcinomas and 1 (3%) was a squamous cell carcinoma. All mutations were confirmed by DNA sequencing. Using 10 ng of starting DNA, the biochip assay demonstrated a detection limit of 1% mutant sequence in a background of wild-type DNA. Our results suggest that the biochip assay is a sensitive alternative to protocols currently in use for KRAS mutation testing on limited quantity samples

KRAS mutation analysis in ovarian samples using a high sensitivity biochip assay

<p>Abstract</p> <p>Background</p> <p>Mutations in the <it>KRAS </it>gene are one of the most frequent genetic abnormalities in ovarian carcinoma. They are of renewed interest as new epidermal growth factor receptor (EGFR)-targeted therapies are being investigated for use in ovarian carcinoma. As <it>KRAS </it>mutations are associated with poor response and resistance to EGFR-targeting drugs, this study was conducted to obtain more information on the spectrum of <it>KRAS </it>mutations in ovarian carcinoma.</p> <p>Methods</p> <p>The presence of <it>KRAS </it>mutations in codon 12 and 13 was analyzed in frozen and formalin-fixed paraffin-embedded (FFPE) tissue with a low density biochip platform. 381 malignant (29 borderline malignancy, 270 primary carcinomas, and 82 recurrent carcinomas) and 22 benign tissue samples from a total of 394 patients were examined. <it>KRAS </it>mutational status of each sample was correlated with dignity, FIGO stage, grade, histology, and survival.</p> <p>Results</p> <p><it>KRAS </it>mutations were found in 60 (15%) samples with 58 samples deriving from malignant tissue and 2 samples deriving from benign tissue. In 55 (92%) samples codon 12 was found to be mutated. Frozen and FFPE samples concurred with respect to <it>KRAS </it>mutation status.</p> <p>Conclusion</p> <p><it>KRAS </it>mutation is a common event in ovarian cancer primarily in carcinomas of lower grade, lower FIGO stage, and mucinous histotype. The <it>KRAS </it>mutational status is no prognostic factor for patients treated with standard therapy. However, in line with experience from colorectal cancer and non-small-cell-lung cancer (NSCLC), it may be important for prediction of response to EGFR-targeted therapies.</p

Method evaluation study of a new generation of vitamin D assays

Introduction: Recently several diagnostic manufacturers have launched new 25-hydroxy-vitamin D (25[OH]D) assays, which are aligned to the National Institute of Standards and Technology (NIST) Standard Reference Materials (SRM) (NIST, Gaithersburg, Maryland). The aim of this study was to compare the performance of one liquid chromatography-tandem mass spectrometry (LC-MS/MS) method, one enzyme linked immunosorbent assay (ELISA), and one recalibrated and previous version of a chemiluminescence immunoassay (CLIA). Material and methods: Serum-aliquots of 198 patient samples from routine 25(OH)D analysis were measured by the ClinMass® LC-MS/MS Complete Kit (RECIPE Chemicals + Instruments GmbH, Munich, Germany), the ORGENTEC 25(OH)D3/D2 ELISA (ORGENTEC Diagnostika GmbH, Mainz, Germany), the recalibrated Immunodiagnostic Systems (IDS)-iSYS 25(OH)DS and the previous used IDS-iSYS 25(OH)D CLIA (Immunodiagnostic Systems Ltd, Boldon, United Kingdom). Bland-Altman and Deming regression analyses were calculated for methods comparison of all tested 25(OH)D assays. The LC-MS/MS method was defined as the reference method. Within-run and between-run precision measurements were performed for all methods with three different concentration levels. Results: Compared to the LC-MS/MS method, the new IDS-iSYS 25(OH)DS and ORGENTEC 25(OH)D3/D2 assay demonstrated mean relative biases of 16.3% and 17.8%. The IDS-iSYS 25(OH)D assay showed the lowest mean bias of 1.5%. Deming regression analyses of the recalibrated IDS-iSYS 25(OH)DS and the ORGENTEC 25(OH)D3/D2 assay showed proportional differences, when compared to the reference method. All assays showed a within-run and between-run imprecision of ≤ 20% at each of the evaluated concentration levels. Conclusions: The evaluated standardized immunoassays and LC-MS/MS are useful methods for measuring 25(OH)D serum-levels in clinical laboratories

Электропривод переменного тока насоса Д200/36 подачи питьевой воды

Цель выпускной квалификационной работы - проектирование асинхронного электропривода центробежного насоса. Выпускная квалификационная работа выполнена с помощью программ MATLAB, Mathcad 14, MS Excel в текстовом редакторе MS Word и представлена на компакт - диске (в конверте на обороте обложки).The purpose of final qualifying work is the design of the asynchronous electric drive of centrifugal pump.Final qualifying work is done using MATLAB software, Mathcad 14, MS Excel to MS Word text editor and presented on the CD - ROM (in an envelope on the back cover)

Temporal Association between Hampton’s Hump Pulmonary Embolism and First-Dose ChAdOx1 nCov-19 Vaccine in a Patient with Activated Protein C Resistance

A 58-year-old man presented to his practitioner with right-sided pleuritic chest pain, dyspnea, and fatigue 18 days following the first dose of the ChAdOx1 nCov-19 vaccine. Chest radiography showed a basal wedge-shaped consolidation indicative of a Hampton’s hump in the right lower lobe, which was confirmed by subsequent computed tomography pulmonary angiography. The major laboratory abnormalities were a markedly elevated D-dimer level of 7.53 µg/mL (normal range Leiden mutation. Neither other errors of hemostasis nor antibodies against platelet factor 4-polyanion complexes could be observed. Moreover, we failed to demonstrate Severe Acute Respiratory Syndrome Coronavirus-2 infection. The patient fully recovered with no sequelae but is being continued on long-term anticoagulation given his risk of recurrent venous thromboembolism. Here we report on the temporal association between the first dose of the ChAdOx1 nCov-19 vaccine and extensive pulmonary embolism in an otherwise healthy patient with activated protein C resistance, however, further investigation is needed to prove causality

Denatured virion protein 1 of equine rhinitis B virus 1 contains authentic B-cell epitopes recognised in an enzyme-linked immunosorbent assay—Short communication

Equine rhinitis B virus 1 (ERBV1), genus Erbovirus, family Picornaviridae , is a pathogen of horses which causes clinical and subclinical infection of the upper respiratory tract in horses. The virus is widespread in European horse populations and the current standard method for the detection of antibody against ERBV1 is by virus neutralisation (VN). VN tests, however, are labour-intensive and time-consuming, require tissue culture facilities, and generally do not provide same-day results. In this study, a protocol for the high-level expression and purification of recombinant virion protein 1 (rVP1) was established using metal-chelate affinity chromatography under denaturing condition. When used as a coating antigen in a prototype enzyme-linked immunosorbent assay (ELISA), denatured rVP1 was recognised by ERBV1 antibody present in horse serum. This finding suggests that denatured rVP1 is a promising candidate for the development of an ELISA to be used in the routine laboratory diagnosis of ERBV1 infection in horses

Refining small intestinal bacterial overgrowth diagnosis by means of carbohydrate specificity: a proof-of-concept study

Background: Diagnosis of small intestinal bacterial overgrowth (SIBO) remains challenging. This study aimed at proving the diagnostic concept of carbohydrate-specific SIBO (cs-SIBO) using glucose, fructose and sorbitol hydrogen (H 2 )/methane (CH 4 ) breath testing (HMBT). Methods: In this study 125 patients referred to our outpatient clinic for SIBO testing were included. All individuals underwent glucose (50 g), fructose (25 g) and sorbitol (12.5 g) HMBT at 3 consecutive days. Patients with cs-SIBO (i.e. early H 2 /CH 4 peak) were given rifaximin (600 mg/day) in a 10-day treatment. HMBT results were reassessed in a subset of patients 3–6 months after antibiotic therapy. In view of cs-SIBO diagnosis, agreements between HMBT results obtained for different sugars were calculated using Cohen’s kappa (κ) with 95% confidence intervals (CIs). Results: A total of 59 (47.2%) patients presented an early H 2 /CH 4 peak with one or more sugars. Among these, 21 (16.8%), 10 (8.0%) and 7 (5.6%) individuals had a positive HMBT result with either glucose, fructose or sorbitol, respectively. Another 21 (16.8%) patients with a positive glucose HMBT result were also found positive with an early H 2 /CH 4 peak obtained after ingestion of fructose and/or sorbitol. Fair agreement was observed between glucose and fructose (κ = 0.26, p = 0.0018) and between glucose and sorbitol (κ = 0.18, p = 0.0178) HMBT results. Slight agreement was observed between fructose and sorbitol (κ = 0.03, p = 0.6955) HMBT results only. Successful antibiotic therapy with rifaximin could be demonstrated in 26/30 (86.7%) of patients as indicated by normal HMBT results and symptom remission. Conclusions: Combined glucose, fructose and sorbitol HMBT has the potential to optimize cs-SIBO diagnosis. Furthermore, the majority of patients with cs-SIBO seem to benefit from rifaximin therapy regardless of its carbohydrate specificity