Introduction to new guidelines for validation of methods to examine visually recognisable substances.

Visual examination of visually recognisable substances, including microscopy, focus on targets or contaminants such as particles of animal origin, plant seeds, spore bodies of moulds, sclerotia, packaging material, microplastic and 'Besatz' (everything that differs from the norm). The two principal results are counts (numbers) and weights for macroscopic methods, or presence/absence for microscopic methods. The level of detection equals at least the size of one unit, usually with a weight exceeding 1 mg, which is in the range of parts per million (ppm). These parameters do not follow a normal distribution but Poisson (counts), lognormal (weights) or binomial (Booleans) distributions, with effect on the interpretation of validation parameters. As for other domains, examination methods for visual monitoring need to be properly validated and quality control during actual application is needed. In most cases procedures for validation of visual methods are based on principles adopted from other domains, such as chemical analysis. A series of examples from publications show inconsistent or not correct implementations of these validation procedures, which stress the need for dedicated validation procedures. Identification of legal ingredients and composition analysis in the domain of visual examination relies on the expertise of the laboratory staff, therefore validation of a method usually includes the validation of the expert. In the view of these specific circumstances, a Guidance for quality assurance and control of visual methods has been developed, which are being presented and discussed in this paper. The general framework of the Guidance is adopted from ISO standards (17023, 17043, 13528). Part 1 of the Guidance includes the general background, theory and principles. Part 2 presents the actual validation procedures with experimental designs and equations for calculating the relevant parameters, and can be used as blueprint for a SOP in a quality management system. An EURL and NRL network for physical hazards is strongly recommended.

Collaborative study on the effect of grinding on the detection of bones from processed animal proteins in feed by light microscopy.

Bone fragments are essential structures for the detection of processed animal proteins (PAPs) in feed by light microscopy for official controls according to Annex VI of European Union Regulation EC/152/2009. The preparation of samples submitted for analysis requires a grinding step to make them suitable for microscopic slide preparation and observation. However, there are no technical guidelines set down for this step despite the fact that it can lead to an increase in bone numbers due to fragmentation. This was demonstrated by an in-house study carried out by the Irish National Reference Laboratory (NRL) for animal protein detection. The present collaborative study investigated the possible effects of three different grinding conditions on the final result for a feed adulterated with 0.05 and 0.01% (w/w) of PAP. The microscopic analysis either combined or not with an Alizarin Red staining was carried out by 10 different laboratories. The results demonstrated that although a large variation in the numbers of bone fragments was noted, five of the six different grinding/staining combinations applied at two levels of PAP adulteration did not significantly (at p = 0.05) differ from one another. The only exception occurred when grinding the feed containing 0.05% of PAP with a rotor mill equipped with a 0.5-mm sieve and combined with a staining which resulted in a greater number of bone fragments by forced fragmentation. Overall, the impact of the grinding/staining combinations on the final results was shown to be negligible when considering the regulatory limit of detection (LOD) requirement for the method and the current rules of implementation of the light microscopic method. From a total of 180 analyses carried out on the feed matrix containing 0.05% of PAP no false-negative result was observed, and at a level of 0.01% PAP only 10 false-negative results occurred.

An Arabidopsis porB porC double mutant lacking light-dependent NADPH:protochlorophyllide oxidoreductases B and C is highly chlorophyll-deficient and developmentally arrested.

A key reaction in the biosynthesis of chlorophylls (Chls) a and b from cyanobacteria through higher plants is the strictly light-dependent reduction of protochlorophyllide (Pchlide) a to chlorophyllide (Chlide) a. Angiosperms, unlike other photosynthetic organisms, rely exclusively upon this mechanism to reduce Pchlide and hence require light to green. In Arabidopsis, light-dependent Pchlide reduction is mediated by three structurally related but differentially regulated NADPH:Pchlide oxidoreductases, denoted as PORA, PORB, and PORC. The PORA and PORB genes, but not PORC, are strongly expressed early in seedling development. In contrast, expression of PORB and PORC, but not PORA, is observed in older seedlings and adult plants. We have tested the hypothesis that PORB and PORC govern light-dependent Chl biosynthesis throughout most of the plant development by identifying porB and porC mutants of Arabidopsis, the first higher plant por mutants characterized. The porB-1 and porC-1 mutants lack the respective POR transcripts and specific POR isoforms because of the interruption of the corresponding genes by a derivative of the maize Dissociation (Ds) transposable element. Single por mutants, grown photoperiodically, display no obvious phenotypes at the whole plant or chloroplast ultrastructural levels, although the porB-1 mutant has less extensive etioplast inner membranes. However, a light-grown porB-1 porC-1 double mutant develops a seedling-lethal xantha phenotype at the cotyledon stage, contains only small amounts of Chl a, and possesses chloroplasts with mostly unstacked thylakoid membranes. PORB and PORC thus seem to play redundant roles in maintaining light-dependent Chl biosynthesis in green plants, and are together essential for growth and development.