EATB Donor Case Workshop 2007.

The European Association of Tissue Banks (EATB) Donor Case Workshop is a forum held within the programme of the EATB annual Congress since 2003. This workshop has been used to discuss clinical donor cases with peer review of practice. It was agreed in advance that the experience of the 2007 workshop should be shared by publication as an example of participative learning which can be extended to other fields within tissue banking and which may be applicable in other disciplines. The EATB Congress in 2008 will extend the idea of participative open workshops with two additional workshops, one on Quality System cases and another on heart valve cases.

Short- and long-term bacterial inhibiting effect of high concentrations of glycerol used in the preservation of skin allografts.

Human skin allografts are important in the treatment of severe burns. Transplantation of skin allografts can cause bacterial transmission. Glycerol in higher concentrations is an appropriate storage medium for allograft cadaver skin and has been attributed an antimicrobial effect. We investigated this effect in more detail. First, the minimal inhibitory concentration of glycerol was determined for 13 bacteria and 1 yeast. This gives an indication about an immediate (20h of incubation) antibacterial effect of glycerol. Second, effect of glycerol in the long-term was studied. Therefore, the survival time was determined for 11 different bacteria suspended in different concentrations of glycerol (50% and 85%) and incubated at three temperatures (4, 24, and 36 degrees C). The minimal inhibitory concentration exceeded 256microg/mL, thus glycerol had no direct inhibitory effect. In contrast, a long-term antimicrobial effect was present and more pronounced at higher concentrations of glycerol and higher temperatures of incubation. The mean survival time of Pseudomonas aeruginosa strains in glycerol 85% at 24 degrees C was 2.6 days, 14.7 days for the tested staphylococci and 29.6 days for three vegetative Bacillus species. In conclusion, microbial safety of glycerol-preserved skin can be increased by preserving skin allografts for some weeks at room temperature.

Comparison of microbiological culture methods in screening allograft tissue. Swab versus nutrient broth.

In order to reduce the risk of transmission of infectious diseases through transplantation of tissue allografts, one should examine tissues for the presence of microorganisms. However, there are no detailed tissue banking guidelines describing the culture method or incubation time to be used. Therefore, we compared two culture methods--blood agar plate versus Wilkins Chalgren broth--and three incubation times--2, 7 or 14 days for their performance. The ultimate aim is to use the optimal setting as standard operating procedure (SOP) for tissue allograft cultures. From 70 consecutive donors, 919 tissue samples were taken. All 919 tissue samples were incubated on blood agar as well as in Wilkins Chalgren broth for 7 days. 567 of these 919 tissue samples were left to incubate up to 14 days. Wilkins Chalgren broth yielded 24.5% (139/567) positive cultures after 14 days of incubation. This was slightly more than the growth on blood agar after 14 days (22.9%--130/567) (p=n.s.) and significantly more than the growth in Wilkins Chalgren broth after 7 days of incubation (21.9%--124/567) (p<0.05). Based on these results, Wilkins Chalgren broth has been implemented as the SOP. Since the yield of positive cultures increased from 2 to 7 days of incubation in broth (1.8 times) and the variability of species cultured from 7 to 14 days of incubation shifted towards mostly microorganisms known to be common contaminants, we established the cut-off at 7 days of incubation in Wilkins Chalgren broth.

Flow cytometric viability assessment and transmission electron microscopic morphological study of bacteria in glycerol.

Human cadaveric skin allografts are used in the treatment of burns and can be preserved in glycerol at high concentrations. Previously, glycerol has been attributed some antimicrobial effect. In an experimental set-up, we aimed at investigating this effect of prolonged incubation of bacteria in 85% glycerol. Staphylococcus epidermidis, Staphylococcus aureus, Pseudomonas aeruginosa, and Bacillus subtilis were incubated in 85% glycerol. The influence of duration of incubation and temperature on ultrastructure and viability were investigated. Unstressed cultures served as controls. Survival was studied after 24-36 h and 10 days incubation in 85% glycerol at 4 degrees C and 36 degrees C with transmission electron microscopy (TEM) and flow cytometry using viability stains indicating membrane damage (SYTO9, propidium iodide) or esterase activity (carboxyfluorescein diacetate). TEM clearly demonstrated variability in morphological changes of bacteria suggesting different mechanisms of damage. Viability stains supported these findings with faster declining viable cell populations in 85% glycerol at 36 degrees C compared with 4 degrees C. Both methods demonstrated that Gram-negative species were more susceptible than Gram-positive species. In conclusion, 85% glycerol may have some additional antimicrobial effect. Temperature is an important factor herein and Gram-negatives are most susceptible. The latter finding probably reflects the difference in cell wall composition between Gram-positive and Gram-negative bacteria.