In vitro susceptibility of high virulence microorganisms isolated in heart valve banking.

Storage preparation of human heart valves for implants generally includes incubation in an antimicrobial disinfection solution and cryopreservation. Changes in patterns of microorganisms susceptibility to antibiotics is a variable process of that promote its inefficiency. The aim of this study has been an evaluation of in vitro susceptibility of high virulence microorganisms isolated in our tissue bank for 14 years in order to evaluate the efficiency, and to promote changes for further antibiotics mixtures as well. Data presented in this study show that microorganisms isolates in valve banking display susceptibility patterns similar to those shown in other clinical circumstances, and the most commonly used antibiotics regimes are useful to date. An antibiotic cocktail containing aminoglicoside in addition to ciprofloxacin and vancomycin is an efficient mixture to be used in valve banking. Further studies will be necessary for monitoring patterns changes of in vitro susceptibility of microbiological isolates in tissue banking.

Cryopreservation increases apoptosis in human menisci.

PURPOSE: Removal of the meniscus leads to progressive degenerative arthritis of the knee on a long-term basis; therefore, meniscal allograft transplantation has been proposed as an alternative to meniscectomy. Preservation methods are required to build up operational stocks and to provide living grafts of a practical size at the right time for patients. Methods for meniscus preservation have been published, and relevant literature confirms that using standard cryopreservation, the chondrocyte survival in situ is inadequate and extremely variable and the cryoinjury mechanisms are not completely established. The aim of the present study is to further investigate possible cellular injury caused by cryopreservation by analysing apoptosis and ultrastructural damage to menisci. METHODS: Seven human menisci that were cryopreserved by standard method were used. All tissue samples were processed simultaneously for routine light microscopy, scanning and transmission electron microscopy as well as apoptosis assessment by the use of ISOL method. RESULTS: With respect to cellularity, significant differences (P < 0.05) between the fresh (14.6 ± 3.5) (mean ± SD) and cryopreserved menisci (9.2 ± 2.8) (mean ± SD) were observed. Apoptosis using ISOL method was observed in fibrochondrocytes of fresh and cryopreserved menisci. The quantitative analysis revealed significant differences (P < 0.05) between fresh meniscus samples, where the apoptotic index was 0.8 ± 2.3% (mean ± SD), and cryopreserved meniscus samples, where this index was 50 ± 18.1% (mean ± SD). CONCLUSION: The results suggest that apoptosis occurs during meniscus cryopreservation. The major findings of this study are cellular damage in meniscus cryopreservation suggesting apoptosis-mediated cell loss. The findings reported herein encourage to further investigations in preservation procedures to enhance maximum long-term clinical survival.

Microbiological analysis of cryopreserved human heart valves after storage: a survey of 3 banks in Spain.

Several reports have shown liquid nitrogen containers as not being sterile. Microorganism transmission has been observed in different cells and tissues stored under this condition, but there is no data on contamination of stored human valves. We performed a survey on heart valve banking in Spain. Regarding the questionnaire, we have a complete microbiological analysis of 304 thawed tissues prior to implant. In six cases positive culture results were observed. Patient follow-up did not reveal any adverse effects. Although some other possibilities should be stated, contamination of heart valves during storage in liquid nitrogen should be considered as a risk element in tissue banking. Strategies to asses and prevent microbial transmission from liquid nitrogen to heart valve banking ought to be further developed.

Keratocyte injury in human corneas cryopreserved under standard conditions.

This study was conducted to characterize ultrastructural damage to human corneas cryopreserved by a standard protocol. The materials used were seven human corneas that were unsuitable for transplantation due to the presence of positive bacteriological cultures; they were cryopreserved according the standard procedure. After freezing and thawing, samples were obtained for scanning and transmission electron microscopy studies. Marked damage was observed in keratocytes with signs of apoptotic cellular injury. However our observations have shown that apoptosis contribute less significantly than necrosis to cellular death in keratocytes of human corneas and although the control of apoptosis is clearly desirable, in order to improve the success of cryopreserved corneas for transplant, we need to continue our investigation to reduce the effects of the necrotic process.

Evaluation and utility of new CE marked containers (CELLFLEX-MacoPharma) for bone bank.

In order to guarantee the required level of quality for our Bone &Tissue Banking, we evaluated a new CE marked container (CELLFLEX MacoPharma), for packing, transport, processing and storage of bones for therapeutic use. The use of CE marked containers is mandatory for organ and tissue containers (Medical Device Directive 93/42). We carried out a three-phase study: (1)Evaluation, (2) Implementation, (3)Audit The product was evaluated for the following criteria:Dash mechanical resistance, Dash air tightness, Dash fragility, Dash capacity. No damage was observed after the storage period, even after immersion in liquid nitrogen. No breakages were observed after provoked impact tests (pots dropped onto the floor). The pot capacity evaluation showed that the inner pot volume (approximately 500 ml) permits adequate storage of tendons and the majority of bone allografts. In conclusion, this evaluation has shown that the CELLFLEX kit is suitable for long duration preservation of bone allografts even at very low temperature conditions (vapour phase nitrogen). Its format and structure permit preservation of most bone allografts.

Implementation of a Quality Plan (ISO 9002) In a Regional Tissue Bank.

Quality control and standardized preservation methods are essential in the field of transplantation. The International Organization for Standardization (ISO) has established a common set of manufacturing, trade and communications standards that are applicable worldwide and that provide the basis of a quality plan for Tissuebreak Banks.The Sectorial Tissue Banking (STB) of the Regional Blood Transfusion Center (RBTC) of Córdoba (Spain) is a non-profit-making tissue bank, established in 1992 to provide tissues for surgical procedures to the hospitals in a regional area. In 1998, the STB as a part of the RBTC embarked upon the path of becoming ISO-certified: after two years of the implementation of the project, STB attained ISO 9002 certification, thus becoming one of the first tissue banks in Europe to achieve this qualification. In this paper we describe the process of becoming ISO-certified, to demonstrate the positive impact that it has had on our entire organization.The assistance of an outside consultant who provided the necessary information for implementing an ISO quality management system was required. The initial improvement was: a well-defined quality manual to address all elements of the ISO 9002 standard, an improved document control system, detailed standard operating procedures (SOPs) and improved employees training processes. A quality committee team and developed quality indicators were created. The internal quality auditing program was established by the selection of employees from a cross-section of the organization, who were trained in internal auditing processes. A formal corrective action system was developed and implemented to facilitate process improvement. The consultant conducted a pre-certification audit, and one month later the certification audit was performed.In conclusion, the implementation of an ISO quality program in the STB has helped our center to establish a control process in the manufacturing of products and services to meet the expectations of our customers, by providing components and services that comply with the national regulatory standards and requirements.

Characterization of ultrastructural damage of valves cryopreserved under standard conditions.

Cryopreserved allograft valves are increasingly being used as valvular replacements. This study was conducted to characterize the ultrastructural damage on the allograft valves obtained by a current standard protocol of valve procurement, antibiotic exposure, and cryopreservation, as a basis for future studies on allograft valve preservation. Materials used were seven aortic and seven pulmonary fresh porcine valves, which were cryopreserved according to the requirements of the American and European Associations of Tissue Banks. The samples were randomly assigned into four groups: (1) fresh, untreated; (2) fresh, treated with antibiotics for 24 h.; (3) treated with antibiotics and exposed to dimethyl sulfoxide (without freezing); and (4) treated with antibiotics, exposed to dimethyl sulfoxide, and then cryopreserved and stored until the study. All tissue samples were processed simultaneously for routine light microscopy and transmission electron microscopy. Fresh-untreated, antibiotic-treated, and dimethyl sulfoxide-exposed valves showed adequate preservation of cellular components. However, after cryopreservation significant damage was observed in fibroblasts with signs of apoptotic cellular injury. Our observation suggests that apoptosis occurs during valve processing. This apoptotic process may be related to various factors, including chemical injury or hypoxia.

Cryopreservation of human skin with propane-1,2-diol.

The tolerance and cryoprotective effect of propane-1,2-diol on human skin was evaluated using tetrazolium reductase enzyme activity to assess tissue viability. The tolerance of human skin was tested by exposing the skin to increasing concentrations of propane-1,2-diol (10, 20, and 40% v/v), at two permeation temperatures (4 degrees C or room temperature), in a stepwise manner, to reduce osmotically induced changes in cell volume. There was no evidence of specific toxicity attributable to the cryoprotectant during the permeation period, even at a concentration of 40%; however a significant decrease in human skin viability was observed after freezing and thawing with high concentrations of cryoprotectant. The only useful protocol was that employing a low cooling rate (-1 degree C min-1) with 10 or 20% (v/v) propane-1,2-diol at a low permeation temperature. The data suggest that propane-1,2-diol does not produced improved results when compared with other cryoprotectants used for skin cryopreservation.

The effect of amphotericin B on the viability of cryopreserved human skin.

To evaluate whether amphotericin B has a detrimental effect on the viability of cryopreserved skin, human skin samples were incubated during 7 and 24 h in three different media: 0.9% phosphate-buffered saline and Medium 199 with and without 10 micrograms/ml amphotericin B. The viability of the tissue was assessed by measuring the tetrazolium reductase activity before and after cryopreservation. A significant decrease in cryopreserved human skin viability was observed after 7 and 24 h incubation with amphotericin B. These results indicate that in the processing of skin preservation, the beneficial effect of using amphotericin B as an antifungal agent may be reduced by its negative effect on tissue viability.

Cyclosporin A versus methotrexate, followed by rescue with folinic acid as prophylaxis of acute graft-versus-host disease after bone marrow transplantation.

Fifty-seven patients undergoing bone marrow transplantation were randomly assigned to receive either cyclosporin A (CsA, n = 26) or methotrexate, followed by rescue with folinic acid (MTX + FA, n = 31) as prophylaxis for graft-versus-host disease (GVHD). All patients but one receiving CsA had evidence of sustained engraftment, and there was no difference between the two groups on the day in which marrow engraftment was documented. Oropharyngeal mucositis was of similar incidence and severity in the two groups. In contrast, patients receiving CsA showed higher renal and hepatic toxicity rates than those treated with MTX + FA. Severe-to-moderate acute GVHD (grades II-IV) was documented in 12 patients receiving CsA and in 12 treated with MTX + FA. The cumulative incidence of this complication was similar in both groups (46.1% and 38.7%). Similarly, there was no difference in the incidence of chronic GVHD. The leukemic relapse rates were also comparable, as well as the estimated probability of survival, which was 55% in patients treated with MTX + FA and 41% in those who were given CsA. We conclude that MTX + FA is as effective as CsA in the prevention of GVHD, with the additional advantage of reduced renal and hepatic toxicities.

No influence of number of donor CFU-GM on granulocyte recovery in bone marrow transplantation for acute leukemia.

The possible interrelation between infused bone marrow CFU-GM and peripheral granulocyte recovery was studied in 16 patients transplanted for acute leukemia. The influence of several clinical events that could modify the graft fate were also analysed. Our results show that: 1) There was no correlation between the number of infused nucleated cells and granulocyte recovery. 2) There was no correlation between the number of infused CFU-GM and granulocyte regeneration. 3) There were significant differences between the day of engraftment in patients with clinically documented HSV infection compared to patients without infection.

A controlled trial of prophylactic granulocyte transfusions during induction chemotherapy for acute nonlymphoblastic leukemia.

Thirty-five noninfected patients undergoing induction chemotherapy for acute nonlymphoblastic leukemia (ANLL) were randomized to either receive (19 patients) or not receive (16 control patients) prophylactic granulocyte transfusions (PGT) when their granulocyte count fell below 0.5 X 10(9)/1. Both groups received identical anti-infectious and supportive care except for granulocyte transfusions. The authors found a nonstatistically significant decrease of the infection rate in the prophylactic group. However, the bacteriologically documented infections and septicemia incidence was significantly higher in the control than in the prophylactic group (P less than 0.05). In the control group they observed in 8 of 16 cases life-threatening infections in contrast with only 1 case in the prophylactic group (P less than 0.01). A significant reduction of deaths due to infectious causes in the prophylactic versus control group were also found (P less than 0.05). The authors did not find an increase of pneumonia or pulmonary infiltrates in the patients belonging to prophylactic in comparison to control group.

Lack of in vitro colony formation in a patient with severe aplastic anemia after spontaneous autologous hematologic reconstitution.

A case of idiopathic severe aplastic anemia with spontaneous complete remission is described. Hematologic parameters normalized spontaneously 94 days after onset. However, the ability of the patient's bone marrow cells to form granulocytic-macrophagic colony-forming units (CFU-GM) or erythroid burst-forming units (BFU-E) was depressed until the 288th day, in spite of the normalization of blood counts. Incubation of the patient's bone marrow cells with antilymphocytic globulin prior to the culture experiment normalized the number of CFU-GM and BFU-E in correlated studies. Coculture of a target marrow with several concentrations of the patient's lymphocytes or serum resulted in a complete inhibition of CFU-GM. BFU-E growth was inhibited by the patient's lymphocytes, but not by serum, which rather showed burst-promoting activity. The inhibitory effect on target bone marrow persisted for 288 days, when it disappeared concomitantly with the restoration of spontaneous CFU-GM and BFU-E growth.