Results of the clinical donor case and quality system case workshops of the European Association of Tissue Banks annual meeting 2009

The European Association of Tissue Banks (EATB) Donor Case Workshop and Quality System Case workshop are forums held within the program of the EATB Annual Congress. These workshops offer an opportunity to discuss and evaluate approaches taken to challenging situations, regarding donor selection and quality issues, and strengthen the professional tissue banking and regulatory networks across Europe. This report reflects some of the discussion at the congress workshops and also subsequent correspondence between the various individuals who submitted cases for discussion. The cases presented to the workshops demonstrate that the findings, their interpretation, deducted actions and preventive measures in tissue banks are not predictable. The varied responses and lack of consensus corroborate this and clearly indicate that operating procedures cannot comprehensively cover or prepare for all eventualities. For many of the issues raised there is a lack of information in the published literature. The workshops actively engage participants, representing a wide array of international expertise, in an informal, secure and enjoyable setting, which facilitates learning from peers and provides potential solutions to those submitting cases. By publishing a summary of the discussions, we hope to reach a wider audience and to stimulate individuals to undertake full literature reviews or research on some of the discussed subjects.     

<Emphasis Type="Italic">Genista</Emphasis> dwarf shrubs as key structures for a Mediterranean grasshopper species on alluvial gravel banks

Natural floodplains belong to the most species-rich ecosystems worldwide. However, over the last decades there has been a strong decrease in the extent of natural floodplains. As a consequence, the biodiversity of these ecosystems has experienced a dramatic decline. In this study, we investigated the habitat and food preferences of the grasshopper species Chorthippus binotatus on gravel banks of a nearly natural river system in the Spanish Pyrenees. The studied plots on the alluvial gravel banks in the Spanish Pyrenees were characterised by strong differences in habitat structure. However, C. binotatus only occurred in sparsely-vegetated sub-Mediterranean dry grasslands. The abundance of the grasshopper species was also strongly related to habitat structure and cover of the main food source and dominant plant species of these open grasslands, the dwarf shrub Genista scorpius. Concerning habitat structure, the grasshopper species preferred habitats with sparse and low-growing vegetation with a high cover of bare ground and gravel/stones. C. binotatus occupies a very narrow ecological niche within the studied floodplain. It requires sparsely-vegetated gravel banks that ensure sufficient oviposition sites and a favourable ambient temperature for optimal development. G. scorpius dwarf shrubs have to be considered as multi-functional key elements in the habitats of C. binotatus. They offer high-quality food, shelter against predators and allow thermoregulation in a hot and dry Mediterranean environment. For the conservation of C. binotatus, we recommend maintaining and restoring both natural floodplain dynamics as well as traditional grazing systems.     

Confirmation of cadaveric blood sample identity by DNA profiling using Short Tandem Repeat (STR) analysis

Blood samples collected from deceased tissue donors for mandatory transfusion microbiology testing may be taken either at the time of tissue donation, or residual samples may be retrieved from hospital laboratories where they were originally used for ante-mortem tests. In the latter case, sample labelling may not conform to the required standard, which stipulates that three independent identifiers be provided. If no alternative adequately labelled sample is available for testing the donated tissues may have to be discarded, which can adversely affect tissue sufficiency. An alternative method to ensure that the blood sample to be tested is from the intended deceased donor is to confirm the identity of the blood sample by Deoxyribonucleic Nucleic Acid (DNA) Short Tandem Repeats (STR) analysis, then comparing the DNA profile with the DNA from the donated tissues. If the two DNA profiles are identical, probability calculations can demonstrate the chance of the two samples of DNA being from the same or different individuals. The authors have used this approach to salvage deceased tissue donations.     

The effect of anti-beta 43-47 Fab fragments on expression of the polymerization sites in the E domain of fibrinogen

In order to assess the significance of the aminoterminal residues of the B beta chain in expression of polymerization sites of the E domain, we have prepared polyclonal antiserum against a synthetic peptide corresponding to beta 43-47 of human fibrinogen. Affinity purified immunoglobulin IgG and Fab prepared from these antibodies reacted strongly and specifically with the synthetic pentapeptide and intact fibrinogen molecule. This specificity was determined both by radioimmunoassay and Western blot analysis of fibrinogen and its plasmic fragments and described in our previous paper (Cierniewski et al., 1986, Biochim. Biophys. Acta, 884, 594-597). Immunochemically purified anti beta 43-47 antibodies and their Fab fragments were strong inhibitors of the fibrin monomer polymerization. Our results imply that amino acid sequence beta 43-47 recognized by these antibodies may be in a close vicinity to the contact sites of the E domain.     

Effects of α-Synuclein Monomers Administration in the Gigantocellular Reticular Nucleus on Neurotransmission in Mouse Model

The aim of the study was to examine the Braak’s hypothesis to explain the spreading and distribution of the neuropathological changes observed in the course of Parkinson’s disease among ascending neuroanatomical regions. We investigated the neurotransmitter levels (monoamines and amino acid concentration) as well as tyrosine hydroxylase (TH) and transglutaminase-2 (TG2) mRNA expression in the mouse striata (ST) after intracerebral α-synuclein (ASN) administration into gigantocellular reticular nucleus (Gi). Male C57BL/10 Tar mice were used in this study. ASN was administrated by stereotactic injection into Gi area (4 μl; 1 μg/μl) and mice were decapitated after 1, 4 or 12 weeks post injection. The neurotransmitters concentration in ST were evaluated using HPLC detection. TH and TG2 mRNA expression were examined by Real-Time PCR method. At 4 and 12 weeks after ASN administration we observed decrease of DA concentration in ST relative to control groups and we found a significantly higher concentration one of the DA metabolites—DOPAC. At these time points, we also noticed the increase in DA turnover determined as DOPAC/DA ratio. Additionally, at 4 and 12 weeks after ASN injection we noted decreasing of TH mRNA expression. Our findings corresponds with the Braak’s theory about the presence of the first neuropathological changes within brainstem and then with time affecting higher neuroanatomical regions. These results obtained after administration of ASN monomers to the Gi area may be useful to explain the pathogenesis of Parkinson’s disease.

     

Interaction of preservation methods and radiation sterilization in human skin processing,with particular insight on the impact of the final water content and collagen disruption. Part I: process validation,water activity and collagen changes in tissues cryopreserved or processed using 50, 85 or 98% glycerol solutions

Current regulatory requirements demand an in-depth understanding and validation of protocols used in tissue banking. The aim of this work was to characterize the quality of split thickness skin allografts cryopreserved or manufactured using highly concentrated solutions of glycerol (50, 85 or 98%), where tissue water activity (a_w), histology and birefringence changes were chosen as parameters. Consistent a_w outcomes validated the proposed processing protocols. While no significant changes in tissue quality were observed under bright-field microscopy or in collagen birefringence, in-process findings can be harnessed to fine-tune and optimize manufacturing outcomes in particular when further radiation sterilization is considered. Furthermore, exposing the tissues to 85% glycerol seems to derive the most efficient outcomes as far as a_w and control of microbiological growth.     

Microhabitat selection in a grassland butterfly: a trade-off between microclimate and food availability

Understanding the factors that determine habitat quality is vital to ensuring appropriate habitat management. The main objective of this study was to assess the microhabitat preferences of egg-depositing females of the Grizzled Skipper (Pyrgus malvae) in calcareous grasslands of the Diemel Valley (Central Germany) for defining habitat quality. Based on this knowledge, we make management recommendations for the conservation of this threatened species. P. malvae generally preferred open and warm oviposition sites. However, there were considerable differences in the environmental conditions, depending on the selected host plant. On the small Potentilla tabernaemontani plants that grew in sparse vegetation with low-growing turf, mostly only one egg was found per plant. In contrast, occupied Agrimonia eupatoria host plants were larger and more prominent, regularly having more than one egg, and grew at sites with a taller and denser vegetation. The observed oviposition pattern reflects a trade-off between microclimate and food availability: Usually, occupied P. tabernaemontani plants grow under favourable microclimatic conditions. However, during hot years the risk of desiccation is high, leading to food shortage. In contrast, A. eupatoria generally provides more biomass, thrives on deeper soils and the vegetation has a cooler microclimate: hence, food shortage is somewhat unlikely. To meet the described habitat requirements of P. malvae, traditional rough grazing by sheep and goats seemed to be the most appropriate land management strategy. The re-introduction of coppicing in woodlands, particularly adjacent to calcareous grasslands, would also be beneficial.     

What determines the distribution of a flightless bush-cricket (<Emphasis Type="Italic">Metrioptera brachyptera</Emphasis>) in a fragmented landscape?

Based on metapopulation theory, isolation, patch size and habitat quality within patches have recently been identified as the most critical parameters determining the persistence of species. In the special case of flightless and sedentary Orthoptera species, taking into account the low dispersal ability, species survival probably depends more on habitat quality than on isolation. The aim of this study was to document how landscape (patch size, isolation and climate) and microhabitat (vegetation structure, microclimate and land use) factors influence patch occupancy and population densities, respectively, of a flightless bush-cricket (Metrioptera brachyptera) in fragmented calcareous grasslands. In summer 2005 patch occupancy of M. brachyptera was assessed in 68 calcareous grassland patches of the Diemel Valley (central Germany). Among these, 26 patches with 80 plots were selected to characterise M. brachyptera habitats in detail. On each plot, bush-cricket density was sampled in an area of 20 m² using a 0.5 m² box quadrat. At the landscape level (patches) in 46 (68%) of 68 studied calcareous grassland patches M. brachyptera was present. Patch occupancy increased with annual precipitation and patch size but was independent of altitude, annual temperature and isolation. At the microhabitat level (plots), population density of M. brachyptera decreased with land-use intensity and increased with vegetation height. In addition, a high litter accumulation was adverse for M. brachyptera. Given the low explanatory power of isolation for patch occupancy, conservation of flightless and sedentary insects, such as M. brachyptera, should primarily focus on improving habitat quality. For M. brachyptera and other stenotopic calcareous grassland species we therefore recommend traditional rough grazing with sheep, which creates a heterogenous habitat structure and avoids the accumulation of too much litter.     

Production and characteristics of human tissue plasminogen-activator antibodies with region-oriented synthetic peptides.

We selected six peptide sequences as belonging to potential epitopes of tissue plasminogen activator (tPA) using, as the main criterion for their choice, the location of the peptide sequences on the surface of the protein molecule. The six peptides (corresponding to amino acids 4-8, 11-16, 96-101, 272-277, 371-376 and 514-519) were synthesized, coupled to carrier proteins and injected into rabbits. All of these peptides elicited antibodies and 15-75% binding of the corresponding iodinated peptide was obtained with a 1:100 dilution of antiserum. Only two anti-(peptide) sera [anti-(tPA96-101) and anti-(tPA272-277)] reacted with intact tPA and its heavy chain in Western immunoblotting analysis. These two peptides sequences and fragment tPA11-16 appear to be involved in the structure of native antigenic epitopes of tPA, since they were recognized and antibodies present in antisera raised against native tPA. There was no interaction between anti-(tPA4-8) and anti-(tPA371-376) sera with intact one-chain or two-chain tPA. In the case of anti-(tPA4-8) cleavage of one-chain tPA to two-chain tPA and reduction of disulfide bonds exposed this epitope.