Standard protocol for the PIGRET assay, a high-throughput reticulocyte Pig-a assay with an immunomagnetic separation, used in the interlaboratory trial organized by the Mammalian Mutagenicity Study Group of the Japanese Environmental Mutagen and Genome Society

The PIGRET assay is one of the Pig-a assays targeting reticulocytes (RETs), an in vivo genotoxicity evaluation method using flow cytometry with endogenous reporter glycosylphosphatidylinositol anchor protein. The PIGRET assay with RETs selectively enriched with anti-CD71 antibodies has several desirable features: high-throughput assay system, low background frequency of mutant cells, and early detection of mutation. To verify the potential and usefulness of the PIGRET assay for short-term testing, an interlaboratory trial involving 16 laboratories organized by the Mammalian Mutagenicity Study Group of the Japanese Environmental Mutagen and Genome Society was conducted. The collaborating laboratories assessed the mutagenicities of a total of 24 chemicals in rats using a single-treatment design and standard protocols for conducting the Pig-a assay on the total red blood cell assay and the PIGRET assay. Here the standard protocol for the PIGRET assay was described in detail.

Evaluierung von Endotoxinproben – Vergleich von Analysen sowie Transport- und Lagerungsbedingungen durch standardisierte Proben/Production of standardized endotoxin dust samples for comparison of transport, storage and analysis of workplace relevant samples

Die Inhalation von Endotoxinen in hohen Konzentrationen kann zu akuten und chronischen Erkrankungen führen. Deshalb ist vor allem an Arbeitsplätzen die Erhebung und – wenn möglich – die Reduktion der Konzentration wichtig. Für die Sammlung, Verarbeitung (Transport, Lagerung, Extraktion) und Analyse von Endotoxinen aus Luftproben an Arbeitsplätzen gibt es zurzeit jedoch keine umfassenden oder aktuell veröffentlichten Verfahren zu qualitätssichernden Kriterien und Maßnahmen. Das führt zu unterschiedlichen Methoden und nicht vergleichbaren Ergebnissen. In der Qualitätssicherung sind Ringversuche ein wichtiger Bestandteil für die Überwachung der Einhaltung von Standardverfahren bei der Analyse. Um die quantitativen Nachweise von Endotoxinen zu vergleichen und Einflüsse durch Lagerung und Transport zu untersuchen, sind Proben notwendig, die kontrolliert und wiederholbar mit definierten Mengen an endotoxinhaltiger Luft beaufschlagt werden. Das in dieser Publikation beschriebene, entwickelte Prozedere erlaubt die gleichzeitige Sammlung von acht Proben unter kontrollierten Bedingungen in einer Bioaerosoltestkammer. Durch Wiederholungen kann damit eine Vielzahl von einheitlich beaufschlagten Filterproben hergestellt werden. Mithilfe solcher Proben konnten Einflüsse von Lagerungszeit und -bedingungen untersucht werden. In einem anschließenden Testringversuch bestimmten neun Labore die Endotoxinaktivitäten von Proben mit verschiedenen Staubkon- zentrationen. Die Labore verwendeten zur Analyse jeweils das in ihrem Haus etablierte Standardverfahren. Während eine Lagerung bei Raumtemperatur von bis zu 14 Tagen keinen Einfluss auf das Ergebnis hat, konnte eine deutliche Reduktion der gemessenen Endotoxingehalte nach einer Lagerung der Extrakte bei -80 °C ermittelt werden.   SUMMARY The inhalation of high concentrations of endotoxins can lead to acute and chronic diseases. Until now there are no extensive and up-to-date criteria and measures for quality control of sampling, processing (transport, storage, extraction) and analysis of endotoxin containing air samples. For quality control purposes, standardized samples have to be produced in a controlled and reproducible way with defined concentrations or endotoxin containing bioaerosols. In this project we developed a setup to produce eight samples simultaneously in a reproducible way to obtain a high number of uniform filter samples, by the use of a bioaerosol test chamber. Using these samples, we investigated storage and transport conditions. After that in a testrun of an interlaboratory trial endotoxin activity of the test samples was determined by nine laboratories. All participants of this trial used the well established method of their lab for extraction and analysis. Our results show, that storage of dust filter samples up to two weeks on room temperature had no significant reduction, but storage of extracts at -80°C showed clear reduction of measured endotoxin activity.

Towards European harmonisation of contagious equine metritis diagnosis through interlaboratory trials

The performance of culture and PCR methods routinely used to diagnose contagious equine metritis (CEM) was evaluated and compared by two interlaboratory trials involving a total of 24 European laboratories, including 22 National Reference Laboratories for CEM. Samples were swab specimens artificially contaminated with bacteria present in the genital tract of Equidae, some with and some without Taylorella equigenitalis, the causative agent of CEM, and T asinigenitalis, responsible for possible misidentification as T equigenitalis. Throughout both interlaboratory trials, PCR performed better in terms of specificity and sensitivity than the culture method, supporting the assertion that PCR should be accepted for CEM diagnosis. However, the culture performance during the second interlaboratory trial was better than during the first one, suggesting that the expertise of participants improved. This reveals the advantage of regular interlaboratory trials to constantly improve the expertise of laboratories. It also shows the need to develop new culture media that are more selective and/or better geared to the metabolism of T equigenitalis in order to improve the bacteriological diagnosis of CEM.

Interlaboratory Comparison of Methods Determining the Botanical Composition of Animal Feed

A consortium of European enterprises and research institutions has been engaged in the Feed-Code Project with the aim of addressing the requirements stated in European Union Regulation No. 767/2009, concerning market placement and use of feed of known and ascertained botanical composition. Accordingly, an interlaboratory trial was set up to compare the performance of different assays based either on optical microscope or DNA analysis for the qualitative and quantitative identification of the composition of compound animal feeds. A tubulin-based polymorphism method, on which the Feed-Code platform was developed, provided the most accurate results. The present study highlights the need for the performance of ring trials for the determination of the botanical composition of animal feeds and raises an alarm on the actual status of analytical inaccuracy.