The first step upon arrival is verification of the package, labelling and transport conditions of the container and the retrieval form. CV tissue processing, evaluation and preservation is performed in a specialised clean room facility. All steps of the protocol are performed under laminar flow Class A, in Class B environment. After dissection of the heart, the tissues are rinsed and macroscopically evaluated looking for atheroma, calcification, aneurism, fenestrations and petechial presence which may cause the tissue discard. Also any significant finding is recorded. Valvular ring and distal artery diameters are measured using calibrated Hegar dilators as well as the length of the aortic and pulmonary arteries. Moreover, a coaptation test is performed filling the conduit with media and looking for leakage to ensure proper performance of the valve. As part of the preparation of the arteries, fat is removed, an inspection for atheroma, calcification or iatrogenic damage is performed and total length and proximal and distal diameters are measured. A photograph of each tissue is taken.
When there is no discarding feature in the tissue, the validated antibiotic cocktail is prepared containing amikacyn (Normon Laboratories - Spain; 791,301), metronidazole (B. Braun Medical SA- Spain; 600,496), ciprofloxacin (Altan Farmaceuticals, S.A.; 643,494), vancomycin (Lab. Reig Jofre, S.A; 606,390) and amphotericin (Xalabarderfarma - Spain) in RPMI (Roswell Park Memorial Institute Medium, Corning, 15,040 CV). The grafts are immersed for overnight decontamination at room temperature. After decontamination, a macroscopic quality control is performed and the grafts are prepared for cryopreservation in a double cryo-bag with cryoprotectants, namely dimethylsulfoxide (DMSO, WAK CHEMIE, WAK DMSO 10) and human albumin (Grifols, 726,609,670,612) in RPMI 1640. Finally, controlled freezing is carried out into a biological chamber (Carburos Matálicos CM2010) in order to decrease the temperature to − 110 °C. Long term storage of the cryopreserved homografts is performed in liquid nitrogen tanks at − 196 °C for 5 years.
Microbiological samples are taken during preparation of the tissue and packaging steps. Biopsy samples taken from the tissue before packaging are included in thioglycollate tubs (Becton Dickinson, 221,787) for the study of bacteries and fungi. Liquid samples from transport media and preservation media are inoculated in BD BACTEC™ PLUS-Aerobic/F Medium (BD Bioscience, 442,192) and BD BACTEC™-Lytic/10 Anaerobic/F Medium (BD Bioscience, 442,265) for aerobic and anaerobic growth and fungi detection. Sistematically, after dissection all hearts are evaluated macroscopically and histologically by the Anatomical Pathology Service of HCB.
When there is no discarding feature in the tissue, the validated antibiotic cocktail is prepared containing amikacyn (Normon Laboratories - Spain; 791,301), metronidazole (B. Braun Medical SA- Spain; 600,496), ciprofloxacin (Altan Farmaceuticals, S.A.; 643,494), vancomycin (Lab. Reig Jofre, S.A; 606,390) and amphotericin (Xalabarderfarma - Spain) in RPMI (Roswell Park Memorial Institute Medium, Corning, 15,040 CV). The grafts are immersed for overnight decontamination at room temperature. After decontamination, a macroscopic quality control is performed and the grafts are prepared for cryopreservation in a double cryo-bag with cryoprotectants, namely dimethylsulfoxide (DMSO, WAK CHEMIE, WAK DMSO 10) and human albumin (Grifols, 726,609,670,612) in RPMI 1640. Finally, controlled freezing is carried out into a biological chamber (Carburos Matálicos CM2010) in order to decrease the temperature to − 110 °C. Long term storage of the cryopreserved homografts is performed in liquid nitrogen tanks at − 196 °C for 5 years.
Microbiological samples are taken during preparation of the tissue and packaging steps. Biopsy samples taken from the tissue before packaging are included in thioglycollate tubs (Becton Dickinson, 221,787) for the study of bacteries and fungi. Liquid samples from transport media and preservation media are inoculated in BD BACTEC™ PLUS-Aerobic/F Medium (BD Bioscience, 442,192) and BD BACTEC™-Lytic/10 Anaerobic/F Medium (BD Bioscience, 442,265) for aerobic and anaerobic growth and fungi detection. Sistematically, after dissection all hearts are evaluated macroscopically and histologically by the Anatomical Pathology Service of HCB.
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