Jan Schroeter

The processing of tissues and cells for transplant bears a risk of microbial contamination which might cause an infection in the recipient. Tissues and cells are therefore treated with antibiotics to reduce the amount of microorganisms. Microbiological controls have to be simple, fast and sensitive and need to be performed according to the specifications of the European Pharmacopoeia. The presented article describes pragmatic approaches for the validation of such specimens with a residual activity of antibiotics inhibiting microbial growth and with that complicating the interpretation of the results.

Introduction: The traceability of tissue and cells transplants is important to ensure a high level of safety for the recipients. With the final introduction of the Single European Code (SEC) in April 2017 in the EU a consistent system among all member states became mandatory. Methods: The regulations for the SEC on EU and national level were evaluated. An overview on the different parts of the SEC with detailed explanations is given. Our own experiences with the implementation of the SEC in our multi-tissue bank are reported in addition. Results: The implementation of the SEC in our multi-tissue bank could be successfully realized. However, it revealed a number of difficulties, especially the sterile labeling of certain tissue transplants and the complex update of the existing database. Conclusion: The introduction of the SEC has made a contribution to the safety of recipients of tissue and cells transplants through a system of comprehensive and transparent traceability.

To evaluate temporary exposure to hyperthermia for its impact on endothelial cell density of porcine corneas in organ culture medium containing dextran with regards to possible negative influences of high temperatures during the storage and transport of corneal grafts. Four groups of central discs (diameter 8 mm) from the corneas of both eyes in 40 pigs were first organ-cultured (MEM with 6% dextran 500) for 24 h at 32°C. Ten corneas were then exposed to 40°C in group 1, to 42°C in group 2, to 44°C in group 3, and to 50°C in group 4 for 12 h each. The paired corneal discs for all groups were not treated, stored at 32°C and served as controls. After further organ culture of all corneas for 48 h at 32°C to allow regenerative processes, corneal endothelium was stained with Alizarin Red S and examined by light microscopy. The endothelial cell densities were determined on three central images using a system for the automatic estimation of morphometric parameters of corneal endothelium. Exposure for 12 h to 40°C as well as to 42°C induced no endothelial cell loss. Statistical analysis showed no significant difference of the endothelial cell density between corneas exposed to 40°C and 42°C and the control corneas (40°C treatment: 4736 ± 426 cells/mm² and control: 4762 ± 344 cells/mm², p = 0.74; 42°C treatment: 4240 ± 363 cells/mm² and control: 4176 ± 448 cells/mm², p = 0.40). Exposure to 44°C and 50°C lead to total necrosis of the endothelial cell layer. Exposure of organ cultured porcine corneas in dextran containing medium up to 42°C for 12 h does not compromise the endothelial cell density in a clinically relevant manner. Temperatures above 42°C, as it might be the case during transports from the cornea bank to the ophthalmic surgeon, must be strictly avoided as they damage the endothelial cell layer.

Background: Since blood culture bottles are validated by the manufacturer for blood only, an additional validation for the use with fluids of tissue preparations is necessary. Methods: Two 10-ml samples of cornea culture medium, histidine-tryptophan-ketoglutarate (HTK) solution, or Ringer solution at the end of femur head thermo-disinfection were given into blood culture bottles (BD BACTEC^TM Plus Aerobic/F, Anaerobic/F for cornea culture medium and BD BACTEC^TM Standard Aerobic/ Anaerobic for HTK and Ringer solution) and subsequently spiked with 10–100 colony forming units (CFU) of bacteria or fungi (aerobic bacteria: Staphylococcus aureus, Bacillus subtilis, Pseudomonas aeruginosa; anaerobic bacteria: Clostridium sporogenes; fungi: Candida albicans, Aspergillus brasiliensis) according to the European Pharmacopoeia Chapter 2.6.1. Results: All tested bacteria and fungi could be detected in all solutions. All positive and negative controls were tested correctly. Compared to the positive controls, the microbial growth was delayed in the antibiotic-containing cornea culture medium, and negative in two cases of B. subtilis spiking. Conclusion: The use of BACTEC^TM blood culture bottles seems to be a suitable method for microbiological testing of HTK solution, Ringer solution, and, with limitations, also for testing of the antibiotic-containing cornea culture medium.

Background: Corneas needed for keratoplasty can be harvested using two techniques: whole globe enucleation and in situ excision of the corneoscleral disc. This study evaluates the rate of microbial contamination of the donor cornea organ culture medium according to the method of retrieval. Methods: All donor corneas of our cornea bank received between January 1, 2001 and December 31, 2009 put into organ culture and microbiologically tested were prospectively analyzed for microbial contamination of the organ culture medium. Results: 2,805 donor corneas could be included in this study in total. 975 of them were retrieved by whole globe enucleation (group 1) and 1,830 by in situ corneoscleral disc excision (group 2). 15 corneas of group 1 (1.5%) and 46 corneas of group 2 (2.5%) showed a contamination of the organ culture medium. The difference was shown not to be statistically significant (p = 0.082). Conclusion: The rate of microbial contamination in organ-cultured donor corneas does not seem to be dependent on the method of their retrieval.

The light microscope is the first-choice technique for the evaluation of organ-cultured donor corneas. For the microscopic visualization of the endothelial cells, the corneas have to be immersed in ahypotonic solution. The number of cells, their vitality and morphology are analyzed. The cell densityis easily estimated with the fixed frame/L method. Use of an image analysis system enables computer processing and counting of digitalized endothelial cell images. An adequately high endothelial cell density after the culture period is a decisive criterion that must be met before releasing adonor cornea for grafting. An endothelial cell density of 2,000-2,200 cells/mm2 is generally recognized as the lower limit for the longest possible graft survival. While an evaluation of the endothelium does not necessarily have to be performed at the beginning or middle of organ culture, it isobligatory at the end. Morphology assessment should routinely involve estimating the pleomorphism (deviation from hexagonality), polymegathism (variation in cell area) and granulation/vacuolization of the endothelial cells. Recognition of devitalized cells is easily facilitated by vital stainingwith trypan blue.

Das neue Gewebegesetz als Folge der Umsetzung der Richtlinien des EU-Parlaments und des Rates 2004/23/EG, 2006/17/EG und 2006/86/EG wird an den Prozessen in der Hornhautbank nichts grundlegend ändern. Es ist aber eine zunehmende Transparenz bei der Gewinnung und Prozessierung von Spenderhornhäuten, eine Harmonisierung bestimmter Arbeitsschritte und ein erhöhtes Bewusstsein für die Bedeutung validierter Abläufe zu erwarten. Bei der Umsetzung des Gesetzes müssen die Besonderheiten der humanen Augenhornhaut berücksichtigt werden, insbesondere ihre natürliche Unsterilität, fehlende Sterilisierbarkeit, postmortale Explantationszeit bis zu 72 h und ihre Transplantierbarkeit trotz Karzinomerkrankung und bakterieller Sepsis des Spenders. Der Tätigkeitsschwerpunkt der Hornhautbanken bleibt die Bereitstellung von Hornhauttransplantaten mit normaler physiologischer Funktionalität und minimalem Risiko einer Krankheitsübertragung. Die Steigerung der Anzahl transplantierbarer Spenderhornhäute ist zur Überwindung des weiterhin bestehenden Mangels dabei eine wesentliche Aufgabe.