Cleanrooms and tissue banking how happy I could be with either GMP or GTP?

The regulatory framework of tissue banking introduces a number of requirements for monitoring cleanrooms for processing tissue or cell grafts. Although a number of requirements were clearly defined, some requirements are open for interpretation. This study aims to contribute to the interpretation of GMP or GTP guidelines for tissue banking. Based on the experience of the participating centers, the results of the monitoring program were evaluated to determine the feasibility of a cleanroom in tissue banking and the monitoring program. Also the microbial efficacy of a laminar airflow cabinet and an incubator in a cleanroom environment was evaluated. This study indicated that a monitoring program of a cleanroom at rest in combination with (final) product testing is a feasible approach. Although no statistical significance (0.90 < p < 0.95) was found there is a strong indication that a Grade D environment is not the ideal background environment for a Grade A obtained through a laminar airflow cabinet. The microbial contamination of an incubator in a cleanroom is limited but requires closed containers for tissue and cell products.

Belgian and European experience with the European Homograft Bank (EHB) cryopreserved allograft valves.--assessment of a 20 year activity.

European Homograft Bank (EHB) has been selecting, preparing, storing and distributing the cryopreserved allograft valves in Belgium and some other European Countries since 1989. It was established in 1988 by a pathologist and the cardiac and vascular surgeons from Belgian and other European centres as an inter-university, international nonprofit association. Due to its neutral behavior and very high quality criteria, European Homograft Bank became one of the prominent heart valve banks in Europe and wider. It collaborates with the transplant coordination in donor selection as well as with the huge network of the implanting surgeons in Belgium and other European Countries. The EHB responsible discusses with the implanting surgeon the allograft selection on basis of the indication and the patients state of emergency. A total of 8.911 donor heart valves have been evaluated in EHB during the last 20 years. After selection, 5.258 allograft valves (1.996 aortic, 3.189 pulmonary and 73 mitral) were cryopreserved and stored in vapors of liquid nitrogen between 6 weeks and 5 years. A total of 4.516 allograft valves (1.391 aortic, 2.620 pulmonary and 48 mitral) were implanted in the left or right ventricular outflow tract for replacement of the diseased aortic or pulmonary valve and for mitral or tricuspid valve replacement or repair. In 1.380 cases the allograft valves were used for right ventricular outflow tract reconstruction as part of the Ross- procedure, whereas in 668 cases the allograft valve served for replacement of the aortic valve for endocarditis. The most important indications for use of cryopreserved allograft valves were: important cardiac and valve malformation in children, female patients of child-bearing age with diseased cardiac valves, cases with contra-indication for anti-coagulation and the patients with severe endocarditis with septal or annular abscesses. Although the number of the donation increased by year, the available allograft valves in stock are still insufficient to respond to all the surgeons' request for different indications.

Access to bacteriophage therapy: discouraging experiences from the human cell and tissue legal framework.

Cultures of human epithelial cells (keratinocytes) are used as an additional surgical tool to treat critically burnt patients. Initially, the production environment of keratinocyte grafts was regulated exclusively by national regulations. In 2004, the European Tissues and Cells Directive 2004/23/EC (transposed into Belgian Law) imposed requirements that resulted in increased production costs and no significant increase in quality and/or safety. In 2007, Europe published Regulation (EC) No. 1394/2007 on Advanced Therapy Medicinal Products. Overnight, cultured keratinocytes became (arguably) 'Advanced' Therapy Medicinal Products to be produced as human medicinal products. The practical impact of these amendments was (and still is) considerable. A similar development appears imminent in bacteriophage therapy. Bacteriophages are bacterial viruses that can be used for tackling the problem of bacterial resistance development to antibiotics. Therapeutic natural bacteriophages have been in clinical use for almost 100 years. Regulators today are framing the (re-)introduction of (natural) bacteriophage therapy into 'modern western' medicine as biological medicinal products, also subject to stringent regulatory medicinal products requirements. In this paper, we look back on a century of bacteriophage therapy to make the case that therapeutic natural bacteriophages should not be classified under the medicinal product regulatory frames as they exist today. It is our call to authorities to not repeat the mistake of the past.

Macrophages and tumor necrosis factor alpha in toxic epidermal necrolysis.

BACKGROUND: We studied the immunopathologic characteristics of five cases of toxic epidermal necrolysis by using a large panel of antibodies. OBSERVATIONS: The pattern and amount of the inflammatory cell infiltrate varied according to the stage of the disease. The main constant feature was the prominent involvement of the monocyte-macrophage lineage, including factor XIIIa+HLA-DR+ dendrocytes and CD68+ Mac 387+ macrophages, before and during the epidermal necrosis. The number of CD4+ and CD8+ lymphocytes was comparatively small. This was associated with a dense labeling of the epidermis for tumor necrosis factor alpha. CONCLUSIONS: Cells of the monocyte-macrophage lineage largely outnumber lymphocytes in the lesions of toxic epidermal necrolysis. Tumor necrosis factor alpha is likely a major cytokine that is responsible for necrosis.

Hyperbaric oxygen therapy and piracetam decrease the early extension of deep partial-thickness burns.

During the first 24 h, a progression of the burn wound in histological depth or extension is often noted. This can only partially be prevented by the routinely used protocols of fluid resuscitation and burn wound dressing. In a rat model of 5% TBSA burn, hyperbaric oxygen therapy (HBOT) and piracetam were evaluated for their ability to further prevent this early deepening of the burn wound. After infliction of the burn wound, the animals were treated with an accepted basic burn wound treatment consisting of mafenide 10% solution humid dressings. They were then randomized into three groups: a control group (n = 10), receiving no other treatment, a HBOT group (n = 17), receiving 60 min of HBOT (203 kPa) twice daily, and a piracetam group (n = 19), receiving piracetam (200 mg/kg IM) twice daily. On the third day of treatment, the entire burn wound was exised and examined histologically. It was found that both HBOT and piracetam had statistically significant effects on the preservation of epidermal basal membrane (P < 0.001 and P < 0.01, respectively). HBOT, but not piracetam, further had significant effects on the destruction of skin appendages (P < or = 0.05 and P > 0.05, respectively) and on the degree of subepidermal inflammation, as measured by leucocyte infiltration (P < 0.001 and P > 0.05, respectively). Furthermore, the HBOT group showed significantly less leucocyte infiltration than the piracetam group (P < 0.01). It was concluded that, although the clinical importance of the small effects on skin appendage and basal membrane preservation may be questionable, the effect on subepidermal leucocyte infiltration is striking and warrants further investigation of the anti-inflammatory effects of HBOT and possibly piracetam.

Burns induced by plants.

Plants of the family Umbelliferae are known to cause a phytophotodermatitis due to a phototoxic reaction caused by furocoumarin (psoralens) and simultaneous exposure to sunlight. We report four patients with partial skin thickness burns, induced by this phototoxic reaction. One occurred after contact with parsley (Apium petroselinum) and three others after contact with giant hogweed (Heracleum mantegazzianum) and simultaneous exposure to sunlight.

HIV transmission by transplantation of allograft skin: a review of the literature.

The fear of human immunodeficiency virus (HIV) transmission by means of allograft skin has led to a cautious approach to allograft donor selection. However, no irrefutable diagnostic test exists to determine the possible presence of HIV at the time of donation. In order to find ways of improving HIV donor screening practices for skin banks, we review the presence of HIV in human skin, explore the possible transmission of HIV by transplantation of human allograft skin, and discuss the reliability of existing HIV tests. The use of the polymerase chain reaction (PCR) as a sensitive detection system for HIV infection of skin biopsies, in combination with conventional routine HIV blood screening tests; could lower the risk of transmitting HIV to severely burned patients.

High frequency percussive ventilation in burn patients: hemodynamics and gas exchange.

High frequency percussive ventilation (HFPV) is a recent ventilatory mode, which combines conventional cycles with high frequency percussions. HFPV was initially instituted as salvage therapy after acute respiratory failure following smoke inhalation injury achieving in each case a dramatic improvement of blood oxygenation, PaCO(2) and ventilatory pressures. This study investigates the influence of HFPV on hesmodynamics, blood oxygenation and ventilatory parameters in eight stable ICU burn patients requiring artificial ventilatory support during a postoperative period following traumatic injury. Periods of 2h were analysed receiving conventional ventilation and HFPV with a high frequency of 400 and 800 cycles/min. Hemodynamic data were not significantly modified; peak inspiratory pressure was significantly lower under HFPV but mean airway pressure was unchanged. Blood oxygenation and CO(2) elimination were significantly improved under HFPV. No side effects were noted. These observations suggest that HFPV could improve pulmonary gas exchanges under lower peak pressures and without hemodynamic compromise. HFPV could represent an interesting alternative open lung strategy method to improve alveolar recruitment.

The usefulness of combined high-frequency percussive ventilation during acute respiratory failure after smoke inhalation.

Inhalation injury and bacterial pneumonia represent some of the most important causes of mortality in burn patients. We describe 11 severely burned patients with acute respiratory failure due to inhalation injury who did not respond adequately to conventional respiratory support. High-frequency percussive ventilation (HFPV) is a recent ventilatory mode, which combines the advantages of conventional ventilation with some of those of high-frequency ventilation. Seven patients developed pulmonary infection during the acute phase; one patient died of multiple organ failure on day 25. All the other patients survived; two developed bronchiolitis obliterans symptoms before discharge. No side-effects were noted and haemodynamic tolerance of HFPV was excellent. Our findings suggest that HFPV can improve pulmonary function and gas exchange in these catastrophic pulmonary failures following inhalation injury.

Psychological approaches during dressing changes of burned patients: a prospective randomised study comparing hypnosis against stress reducing strategy.

A prospective study was designed to compare two psychological support interventions in controlling peri-dressing change pain and anxiety in severely burned patients. Thirty patients with a total burned surface area of 10-25%, requiring a hospital stay of at least 14 days, were randomised to receive either hypnosis or stress reducing strategies (SRS) adjunctively to routine intramuscular pre-dressing change analgesia and anxiolytic drugs. Visual analogue scale (VAS) scores for anxiety, pain, pain control and satisfaction were recorded at 2-day intervals throughout the 14-day study period, before, during and after dressing changes. The psychological assistance was given on days 8 and 10 after hospital admission. The comparison of the two treatment groups indicated that VAS anxiety scores were significantly decreased before and during dressing changes when the hypnotic technique was used instead of SRS. No difference was observed for pain, pain control and satisfaction, although VAS scores were always better in the hypnosis group. The study also showed that, overall, psychological support interventions reduced pain and increased patient satisfaction. These results confirm the potential benefits of psychological assistance during dressing changes in burned patients.

Medical treatment of a central vein suppurative thrombosis with cerebral metastatic abscesses in a burned child.

A 2-year-old girl admitted with third degree burns (35% TBSA) received 7 weeks poly-antibiotic therapy combined with heparin for a severe Methicillin-resistant Staphylococcus aureus sepsis with multiple metastatic abscesses (lung, skin, brain), from a suppurative thrombophlebitis of the right jugularis interna, extended to the axillary and cava superior veins. Surgical treatment was contraindicated by the local extension. The child was discharged without major neurological sequelae 3 months after admission.

High frequency percussive ventilation and conventional ventilation after smoke inhalation: a randomised study.

Inhalation injury and bacterial pneumonia represent some of the most important causes of mortality in burn patients. Thirty-five severely burned patients were randomised on admission for conventional ventilation (CV; control group) versus high frequency percussive ventilation (HFPV; study group). HFPV is a ventilatory mode, introduced 10 years ago which combines the advantages of CV with some of those of high frequency ventilation. Arterial blood gases, ventilatory and hemodynamic variables were recorded for 5 days at 2h intervals. Incident complications were classically managed. A statistical analysis (Student's t-test and Wilcoxon signed rank test) demonstrated a significant higher PaO(2)/FiO(2) from days 0 to 3 in the HFPV group. No significant differences were observed for the other parameters. Our findings suggest that HFPV can improve blood oxygenation during the acute phase following inhalation injury allowing reduction of FiO(2). No significant differences were observed between groups for mortality nor incidence of infectious complications in this study.

Analysis of epidemic Pseudomonas aeruginosa isolates by isoelectric focusing of pyoverdine and RAPD-PCR: modern tools for an integrated anti-nosocomial infection strategy in burn wound centres.

The Gram-negative bacterium Pseudomonas aeruginosa is an important life-threatening nosocomial pathogen in burn units. In this study we analysed epidemic P. aeruginosa isolates from patients and their hospital environment using two new molecular techniques in order to establish strain relatedness for epidemiological purposes. One technique was pyoverdine typing by isoelectric focusing (PVD-IEF) and the other was a genomic PCR-based fingerprinting technique called random amplification of polymorphic DNA actually referred to as RAPD-PCR. The described short epidemic (6 weeks) included 37 consecutive isolates from 9 different patients as well as two environmental isolates recovered, at the same time, from one of the hydrotherapy facilities. Only two of the three known pyoverdine types of P. aeruginosa could be found. Type I was absent while type II represented 49 per cent and type III, 51 per cent of the isolates. The two consecutive isolates from the environment were both of type III. The RAPD-PCR fingerprinting discriminated four patterns. Profile 1 represented 60 per cent; profile 2, 34 per cent; and profiles 3 and 4 only 3 per cent of the isolates respectively. The environmental isolates also had a RAPD-PCR 1 profile, arguing for the hydrotherapy facility as a possible contamination source. Prompt measures could prevent an outbreak. The study demonstrates the applicability of the techniques in a routine microbiology lab as well as their usefulness, in combination with other techniques, in the fight against nosocomial infections, which are so critical in burn units. Both techniques showed undoubtable evidence of the occurrence of polymicrobial infection of individual patients by P. aeruginosa species. Meanwhile pyoverdine typing by IEF seems suited to studying more profoundly the role of pyoverdines in burns.

Lyophilized keratinocyte cell lysates contain multiple mitogenic activities and stimulate closure of meshed skin autograft-covered burn wounds with efficiency similar to that of fresh allogeneic keratinocyte cultures.

For several years, grafting with allogeneic keratinocyte cultures has been used successfully as a wound-healing therapy both by us and by many other groups. Since their postgrafting survival time is limited, the effect of these cultures is generally explained by the production of wound repair-stimulating factors that promote proliferation and migration of resident cells. In this study we show that lysates of cultured keratinocytes contain mitogenic activity for keratinocytes, endothelial cells, and fibroblasts. In addition, the lysates inhibit the contraction of collagen gels by human skin fibroblasts. On the basis of these observations and of in vivo data obtained by ourselves and others, we have evaluated the effect of total keratinocyte lysates on the healing of meshed skin autograft-covered burn wounds. Twenty burn wounds were tangentially excised and autografted with one to three meshed conventional skin transplants. An area treated with a gel containing lysated keratinocyte cultures was compared with an area treated with placebo-gel in terms of epithelialization on day 5. In six patients an additional fresh keratinocyte alloculture was applied as a positive control. Results indicate that the newly formed epithelium (difference between percentage of epithelialization on day 5 and on day 0) was 31.1 percent in the treated area compared with 16.5 percent in the placebo area. This result is comparable with the value obtained by treatment with fresh keratinocyte allocultures, namely, 33.8 percent. These figures show a twofold stimulation of epithelialization.

Cultured allogeneic keratinocyte sheets accelerate healing compared to Op-site treatment of donor sites in burns.

Donor site treatment is a crucial issue in the treatment of extensive burns. In this single-blind, randomized study treatment of donor sites with a polyurethane dressing, Op-Site (Smith & Nephew, York, U.K.) is compared to treatment with allogeneic cultured keratinocyte sheets. Results show a mean healing time of 6.7 days with use of cultured keratinocyte sheets compared to mean healing time of 13.6 days with Op-Site treatment. Also, improvement in the comfort of patients as the result of less exudate formation and pain attenuation was noted.

Processing of ovine cardiac valve allografts: 1. Effects of preservation method on structure and mechanical properties.

It is essential to have some method of preservation of allograft valves during the time between procurement and implantation. Cryopreservation is the most commonly-used storage method today but it has the major disadvantage of high cost, and because its aim is to preserve living cells only relatively gentle antimicrobial treatments are used. This study addresses two interrelated questions: Is it necessary to maintain living donor cells in the tissue graft? Can more effective measures be used to reduce the risk of transmission of diseases, especially viral diseases, via human tissue grafts. In this paper, we report an investigation of four preservation methods that could be combined with more effective disinfection: cryopreservation with dimethyl sulphoxide, storage at approximately 4 degrees C in a high concentration of glycerol as used for the preservation of skin, snap-freezing by immersion in liquid nitrogen and vitrification. Snap freezing was mechanically damaging and vitrification proved to be impracticable but two methods, cryopreservation and storage in 85% glycerol, were judged worthy of further study. Cryopreservation was shown to maintain cellular viability and excellent microscopic structure with unchanged mechanical properties. The glycerol-preserved valves did not contain any living cells but the connective tissue matrix and mechanical properties were well preserved. The importance of living cells in allograft valves is uncertain. If living cells are unimportant then either method could be combined with more effective disinfection methods: in that case the simplicity and economy of the glycerol method would be advantageous. These questions are addressed in the two later papers in this series.

Processing of ovine cardiac valve allografts: 3. Implantation following antimicrobial treatment and preservation.

It is known that a satisfactory clinical outcome can follow the implantation of cardiac valve allografts in spite of the loss of living cells in the tissue. If viable cells are not required for long term graft function, then effective disinfection of the tissue might become possible. In an earlier paper in this series we reported that peracetic acid (PAA) is an effective antimicrobial agent for the treatment of valve allografts; it was lethal to the cells but at a concentration of 0.21% had little effect on the mechanical properties or extracellular morphology of the valve leaflets. It was also found that PAA-treatment could be combined with storage in 85% glycerol at 4 degrees C, or cryopreservation with 10% Me(2)SO, without substantial further impairment of microscopic structure or mechanical properties. In this paper we describe the implantation of processed ovine aortic valves in the descending thoracic aorta of sheep. The experimental groups included control untreated valves and valves that had been treated with antibiotics or PAA and either cryopreserved, or stored in 85% glycerol. The recipient sheep showed good clinical appearances until the experiment was terminated at six months. The explanted grafts were examined by standard morphological and mechanical testing methods. The PAA-treated valves were clearly recognisable as valves: the leaflets had fair to medium morphology in both the unpreserved and the cryopreserved groups. All leaflets had a superficial overgrowth of cells. Microsatellite analysis for allelic differences were performed on samples of donor and recipient tissues using three markers of tissue source. Only one valve, which had been treated with PAA, revealed allelic differences between donor and recipient. It is suggested that DNA-fragments may have remained after the destruction of donor cells and six months of implantation: the overgrowing cells were almost certainly of recipient origin. We conclude that our experiments, in which PAA-treatment was combined with preservation, are sufficiently encouraging to justify further studies to refine the technique, but in our opinion they are not sufficient to justify a clinical trial at this time.

Enhancement of healing of mesh grafts with autologous keratinocytes.

Cultured epithelium prepared from epidermal cells may spread to cover large wounds and may also stimulate the growth of islands of intact cells. Autologous keratinocytes have been used in two cases to enhance the healing of mesh grafts laid on large surgical wounds.

European homograft bank: twenty years of cardiovascular tissue banking and collaboration with transplant coordination in Europe.

Established in 1989 in Brussels as an international nonprofit association, the European Homograft Bank (EHB) has been collaborating closely with the transplant coordination of the different centers in Belgium and other European countries. Donor selection is made after discussion of exclusion criteria with the transplant coordinator of the procurement center. EHB collaborates with 15 Belgian, 11 German, 10 French, 10 Swiss, 3 Italian, 3 Dutch, and some other procurement and/or implantation centers. Donor ages range from newborn to 65 years. Tissue preparation, morphologic evaluation, and functional testing are performed under Class A laminar flow. After decontamination in a cocktail of 3 antibiotics (lincomycin, vancomycin, and polymixin B) during 20-48 hours, the tissues cryopreserved with liquid nitrogen to -100 degrees C are stored in vapors of liquid nitrogen below -150 degrees C for a maximum of 5 years. Systematic virologic examination of donor blood is performed for HIV, HTLV, hepatitis B/C, and syphilis, as well as for enteroviruses, Q fever, malaria, and West Nile virus by indication. Bacteriologic examination for anaerobic and aerobic contamination is performed at the different steps of processing. Histologic examination for malignant disease and infection is performed systematically. Indications for implantation are discussed with the requesting surgeon. Transport to the implantation center is carried out safely in a dry shipper at -150 degrees C or in dry ice at -76 degrees C. The EHB received 4,511 hearts and 1,169 batches of arteries from January 1989 to December 2008. The 5,133 heart valves (1,974 aortic, 3,106 pulmonary, and 53 mitral) and 2,066 arterial segments have been prepared and stored; 4,600 cryopreserved valvular (2,717 pulmonary, 1,835 aortic, and 48 mitral) and 1,937 arterial allografts have been distributed for implantation in various European Cardiovascular Centers. EHB is not always able to meet the increased demand for heart valves and arterial allografts. Collaboration between the EHB and the Transplant Coordination is satisfactory. Donor selection criteria are discussed with the transplant coordinator; whereas, implantation indication, with the implanting surgeon. Because the EHB is not always able to meet demands for the cryopreserved valves and arterial segments, there is a need to increase number of procurements. Cardiovascular surgeons need to play more active roles in the resolution of this problem.

Decontamination of heart valve and arterial allografts in the European Homograft Bank (EHB): comparison of two different antibiotic cocktails in low temperature conditions.

The aim of the study was to compare the efficiency of two different antibiotic cocktails in the cardiovascular allograft decontamination. Low temperature, low-concentration antibiotic cocktail with Cefoxitin, Lincomycin, Polymixin B and Vancomycin was decontamination protocol in EHB for many years. The modified cocktail doesn't contain Cefoxitin. The study had two steps. First step: cardiovascular allografts from 80 donors are incubated in classical (group 1) or modified cocktail (group 2). Second step: 184 and 182 allografts of group 1 and group 2 are incubated in the modified and classical antibiotic cocktail, respectively. The bacteriological examination is performed in three steps: A-transport solution, B-decontamination solution and C-cryopreservation solution. During the first step 23.75% of the tissues were initially contaminated mainly with Staphylococcus (78.95%). 93.75% of the allografts of group 1 and 100% of group 2 were sterile after incubation (p = 0.058). 25.54% and 30.77% of group 1 and 2, respectively were contaminated in A-examination during the second step. Staphylococci were isolated in 82.98% and 69.64% in group 1 and 2, respectively. About 4.35% of group 1 and 5.5% of group 2 were contaminated in A, B, and C whereas 5.4% of group 1 and 4.4% of group 2 were contaminated in B or C after being sterile in A. Finally 9.78% of the tissues were rejected and 90.22% cryopreserved in the modified, whereas 9.89% rejected and 90.11% accepted in the classical group (p = 0.1). The difference was non-significant in the level of decontamination between the two cocktails. Contamination of some tissues with low growing, low-pathogen germs that appeared in B or C examination, couldn't be explained. This issue needs complementary investigation.