Bacterial contamination of amniotic membrane in a tissue bank from Iran

Human Amniotic Membrane (AM) transplantation can promote tissue healing and reduce inflammation, tissue scarring and neovascularization. Homa Peyvand Tamin (HPT) tissue bank has focused on manufacturing human cell and tissue based products including AM. The purpose of this study is to evaluate and identify bacterial contamination of AMs that is produced by HPT for several ophthalmic applications. From July 2006 to April 2011, 122 placentas from cesarean sections were retrieved by HPT after obtaining informed consent from the donors. Besides testing donor’s blood sample for viral markers, microbiological evaluation was performed pre and post processing. During tissue processing, decontamination was performed by an antibiotic cocktail including; Gentamicin, Ceftriaxone and Cloxacillin. Of 271 cesarean section AM donors who were screened as potential donors, 122 were accepted for processing and assessed for microbiological contamination. Donors’ age were between 21 and 41 years (Mean = 27.61 ± 0.24). More than 92 % of mothers were in their first or second gravidity with full term pregnancies. The most prevalent organisms were Staphylococci species (72.53 %). After processing, contamination rates markedly decreased by 84.62 % (p value = 0.013). According to our results, most of bacterial contaminations were related to donation process and the contamination pattern suggests procurement team as a source. Therefore we recommend that regular training programs should be implemented by tissue banks for procurement staff. These programs should focus on improved donor screening and proper aseptic technique for tissue retrieval. We also suggest that tissue banks should periodically check the rate and types of tissue contaminations. These data help them to find system faults and to update processing methods.     

Analysis of the effectiveness of Sodium Hypochlorite decontamination of cadaveric human tissues at retrieval

Bacterial contamination of tissues retrieved from cadaveric donors is a common feature worldwide, and every tissue bank, albeit using different methods, conducts decontamination to guarantee safe tissues suitable for clinical use. The effectiveness of the methods used to eradicate pathogens differs. In order to reduce the tissue bioburden at retrieval, we have introduced a new method involving rinsing tissues in a sodium hypochlorite solution. To test its effectiveness we analyzed two comparable groups of tissues: Group A: 1881 tissues, all rinsed with isotonic saline solution after retrieval, and Group B: 1968 tissues immersed in an isotonic saline solution containing sodium hypochlorite (final concentration 0.1 %) for different lengths of time and subsequently rinsed with isotonic saline. The rinsing solution of each tissue was then sampled for microbiological cultures in both groups. The resultant overall contamination rate was 40.5 % for Group A and 6.7 % for Group B, with an 82.8 % difference in the reduction of contamination between the two groups. This was especially the case for commensal skin bacteria in musculoskeletal tissue, which accounted for over half the overall contamination. Our data highlighted that decontamination with sodium hypochlorite was helpful in reducing the bacterial bioburden in tissues retrieved from cadaveric donors.     

The effect of sterilization on mechanical properties of soft tissue allografts

One major concern regarding soft tissue allograft use in surgical procedures is the risk of disease transmission. Current techniques of tissue sterilization, such as irradiation have been shown to adversely affect the mechanical properties of soft tissues. Grafts processed using Biocleanse processing (a proprietary technique developed by Regeneration Technologies to sterilize human tissues) will have better biomechanical characteristics than tissues that have been irradiated. Fifteen pairs of cadaveric Achilles tendon allografts were obtained and separated into three groups of 10 each. Three treatment groups were: Biocleanse, Irradiated, and Control (untreated). Each specimen was tested to determine the biomechanical properties of the tissue. Specimens were cyclically preloaded and then loaded to failure in tension. During testing, load, displacement, and optical strain data were captured. Following testing, the cross sectional area of the tendons was determined. Tendons in the control group were found to have a higher extrinsic stiffness (slope of the load–deformation curve, p = .005), have a higher ultimate stress (force/cross sectional area, p = .006) and higher ultimate failure load (p = .003) than irradiated grafts. Biocleanse grafts were also found to be stiffer than irradiated grafts (p = .014) yet were not found to be statistically different from either irradiated or non-irradiated grafts in terms of load to failure. Biocleanse processing seems to be a viable alternative to irradiation for Achilles tendon allografts sterilization in terms of their biomechanical properties.     

Initiation of Bone and Amnion Banking in Turkey

There is a growing demand in Turkey for human tissue to use in surgery and wound healing. However, our country does not have facilities for local production of tissue grafts and generally depends on imported products. Under a multi-year project initiated in 1997, the International Atomic Energy Agency has provided main equipment for tissue processing and experts on Tissue Banking as well as training on tissue processing methods.In this presentation, information on various stages of the project implementation is given. Details of lay out for the process laboratories and equipment are given. Donor selection and testing criteria, processing procedures for bone and amnion, setting up product design, implementation of quality system and radiation sterilisation are described briefly. Quality procedures included preparation of quality manual, record forms, document control, non-conformance and corrective actions, training records, equipment maintenance and calibration are all in line with GMP/GLP Standards. Clinical applications of tissue grafts and medico-legal position of organ and tissue donation in Turkey are also discussed briefly.     

Disinfection of human skin allografts in tissue banking: a systematic review report

The use of skin allografts to temporarily replace lost or damaged skin is practiced worldwide. Naturally occurring contamination can be present on skin or can be introduced at recovery or during processing. This contamination can pose a threat to allograft recipients. Bacterial culture and disinfection of allografts are mandated, but the specific practices and methodologies are not dictated by standards. A systematic review of literature from three databases found 12 research articles that evaluated bioburden reduction processes of skin grafts. The use of broad spectrum antibiotics and antifungal agents was the most frequently identified disinfection method reported demonstrating reductions in contamination rates. It was determined that the greatest reduction in the skin allograft contamination rates utilized 0.1 % peracetic acid or 25 kGy of gamma irradiation at lower temperatures.     

Fetal Tissue Banking for Transplantation: Characteristics of the Donor Population and Considerations for Donor and Tissue Screening

We initiated this study to evaluate the suitability for therapeutic use in transplantation of tissues obtained from human abortuses. We have developed protocols for the collection, handling and preservation of hepatic stem cells from electively aborted embryos and have developed methods for assessment of the cells so derived and processed. In this paper we present our findings regarding screening of potential donors, acquisition of fetal tissues, and assessment of the tissues for potentially infectious contaminants. We assess the suitability of the tissue donors according to current standards used for donors of commonly transplanted tissues (e.g., bone grafts, skin grafts and heart valves) and present data regarding the real availability of tissues from elective abortion procedures that would meet those standard tissue banking criteria.We specifically evaluated the donor's willingness to provide a blood sample for testing, conducted a detailed interview similar to those used for typical organ and tissue donors, and assessed the type and incidence of contamination in collected tissues. We find that although many women are willing to consent to use of the tissues for transplantation, attrition from the study for various reasons results in few fetal organs ultimately realistically available for transplantation. Typical reasons for attrition include: unwillingness to have a blood sample drawn or tested, positive serology results, social/medical high risk factors for acquisition of transmissible disease, no identifiable organs available, and unacceptable microbial contamination. Thus, although it might seem that due to the numbers of abortions performed annually, that there would be substantial numbers of suitable tissues available, only a small proportion are truly suitable for transplantation.     

Advances of radiation sterilisation in tissue banking

Under the auspices of the IAEA tissue banking programme on “Radiation Sterilisation of Tissue Graft” conducted from 1985 to 2004, many scientists and surgeons were involved in various regional research and development (R&D) projects mainly in dealing with radiation dose selection, radiation effects on human tissues and quality system in radiation sterilisation. New findings on radiation effects, tissue processing and preservation were shared during the regional and interregional meetings and workshops. Many tissue banks started to use radiation (25 kGy) to sterilize tissue grafts for tissue safety and efficacy and still continue to use it. The IAEA Code of Practice for Radiation Sterilization of Tissues Allografts developed in 2007 offered simpler methods to conduct radiation dose setting and dose validation experiments for tissue grafts. Advances in dose selection and dose mapping are continued under the quality management system when banks need to be certified to continue their operation. The combination of good tissue processing and preservation as well as good radiation practice will ensure the tissue products are properly sterilised thus safe and of high quality. Experience in meeting challenges in using radiation sterilisation and achievements reported by the tissue bankers are shared here.     

Tissue recovery practices and bioburden: a systematic review

For successful transplantation, allografts should be free of microorganisms that may cause harm to the allograft recipient. Before or during recovery and subsequent processing, tissues can become contaminated. Effective tissue recovery methods, such as minimizing recovery times (<24 h after death) and the number of experienced personnel performing recovery, are examples of factors that can affect the rate of tissue contamination at recovery. Additional factors, such as minimizing the time after asystole to recovery and the total time it takes to perform recovery, the type of recovery site, the efficacy of the skin prep performed immediately prior to recovery of tissue, and certain technical recovery procedures may also result in control of the rate of contamination. Due to the heterogeneity of reported recovery practices and experiences, it cannot be concluded if the use of other barriers and/or hygienic precautions to avoid contamination have had an effect on bioburden detected after tissue recovery. Qualified studies are lacking which indicates a need exists for evidence-based data to support methods that reduce or control bioburden.     

Skin donors and human skin allografts: evaluation of an 11-year practice and discard in a referral tissue bank

The Saint Louis hospital tissue bank provides skin allografts to pediatric and adult burn units in the Paris area. The aim of this study was to analyze our activity during the last 11 years focusing on the reasons for skin discard. Skin is procured solely from the back of the body, which is divided into 10 zones that are harvested and processed separately. This retrospective study included all skin donors harvested between June 2002 and June 2013, representing a total of 336 donors and 2770 zones. The donors were multiorgan heart-beating donors in 91 % of cases (n = 307). The main reason for discarding harvested skin was microbial contamination, detected in 99 donors (29 %). Most contaminants were of low pathogenicity. Other reasons for discard included positive serologic tests for 2 donors [17 zones (0.61 %)], unsuitable physical skin characteristics for 3 zones (0.11 %), the donor’s medical history for 53 zones (1.91 %), and technical issues with processing or distribution for 61 zones (2.2 %). In our experience, microbial contamination continues to be the main reason for discarding potential skin allografts. However, discards are limited by separate harvesting and processing of multiple zones in each donor.     

Validation of an endothelial roll preparation for Descemet Membrane Endothelial Keratoplasty by a cornea bank using “no touch” dissection technique

Descemet Membrane Endothelial Keratoplasty (DMEK) selectively replaces the damaged posterior part of the cornea. However, the DMEK technique relies on a manually-performed dissection that is time-consuming, requires training and presents a potential risk of endothelial graft damages leading to surgery postponement when performed by surgeons in the operative room. To validate precut corneal tissue preparation for DMEK provided by a cornea bank in order to supply a quality and security precut endothelial tissue. The protocol was a technology transfer from the Netherlands Institute for Innovative Ocular Surgery (NIIOS) to Lyon Cornea Bank, after formation in NIIOS to the DMEK “no touch” dissection technique. The technique has been validated in selected conditions (materials, microscope) and after a learning curve, cornea bank technicians prepared endothelial tissue for DMEK. Endothelial cells densities (ECD) were evaluated before and after preparation, after storage and transport to the surgery room. Microbiological and histological controls have been done. Twenty corneas were manually dissected; 18 without tears. Nineteen endothelial grafts formed a double roll. The ECD loss after cutting was 3.3 % (n = 19). After transportation 7 days later, we found an ECD loss of 25 % (n = 12). Three days after cutting and transportation, we found 2.1 % of ECD loss (n = 7). Histology found an endothelial cells monolayer lying on Descemet membrane. The mean thickness was 12 ± 2.2 µm (n = 4). No microbial contamination was found (n = 19). Endothelial roll stability has been validated at 3 days in our cornea bank. Cornea bank technicians trained can deliver to surgeons an ECD controlled, safety and ready to use endothelial tissue, for DMEK by “no touch” technique, allowing time saving, quality and security for surgeons.     

Structural changes of skin and amnion grafts for transplantation purposes following different doses of irradiation

An important part of the preparation of biological material for transplantation is sterilization. The aim of our study was to assess the impact of ionizing radiation on three types of biological tissues and the impact of different doses on cells and extracellular matrix. Three types of frozen tissues (porcine skin xenografts, human skin allografts and human amnion) were divided into five groups, control and groups according to the dose of radiation to which these samples were exposed (12.5, 25, 35 and 50 kGy). The tissue samples were fixed by formalin, processed by routine paraffin technique and stained with hematoxylin and eosin, alcian blue at pH 2.5, orcein, periodic acid schiff reaction and silver impregnation. The staining with hematoxylin and eosin showed hydropic degeneration of the cells of epidermis in xenografts by the dose of 12.5 kGy, in human skin it was observed by the dose of 35 kGy. The staining for elastic fibers revealed damage of fine elastic fibers in the xenografts dermis by the dose of 12.5 kGy, in the allografts by 35 kGy. Another change was the disintegration of basement membrane of epithelium, especially in the human amnion at the dose of 50 kGy. The silver impregnation visualized nuclear chromatin condensation mainly in human amnion at the dose of 12.5 kGy. Our results have shown that the porcine xenografts and human amnion were more sensitive to irradiation than the human skin. In the next phase of the project we will focus at more detailed changes in the tissues using immunohistochemical techniques.     

Effects of ionizing radiation and preservation on biomechanical properties of human costal cartilage

Tissue banks around the world store human cartilage obtained from cadaveric donors for use in diverse reconstructive surgical procedures. To ensure this tissue is sterile at the time of distribution, tissues may be sterilized by ionizing radiation. In this work, we evaluate the physical changes in deep frozen costal cartilage (?70 °C) or costal cartilage preserved in high concentrations of glycerol (>98 %) followed by a terminal sterilization process using ionizing radiation, at 3 different doses (15, 25 and 50 kGy). Tension and compression tests were carried out to determine the mechanical changes related both to the different preservation methods and irradiation doses. For both methods of preservation, tension strength was increased by about 24 %, when cartilage tissue was irradiated with 15 kGy. Deep frozen samples, when irradiated with 25 or 50 kGy, had a decrease in their mechanical performance, albeit to a lesser extent than when tissues were preserved in high concentration of glycerol and equally irradiated. In conclusion, processing in high concentration of glycerol did not increase tissue protection against radiation damage; while cartilage preserved in high concentrations of glycerol withstands radiation up to 25 kGy, deep frozen human costal cartilage may be sterilized with a doses up to 50 kGy without significant mechanical impact.     

The implementation of tissue banking experiences for setting up a cGMP cell manufacturing facility

Cell manufacturing for clinical applications is a unique form of biologics manufacturing that relies on maintenance of stringent work practices designed to ensure product consistency and prevent contamination by microorganisms or by another patient's cells. More extensive, prolonged laboratory processes involve greater risk of complications and possibly adverse events for the recipient, and so the need for control is correspondingly greater. To minimize the associate risks of cell manufacturing adhering to international quality standards is critical. Current good tissue practice (cGTP) and current good manufacturing practice (cGMP) are examples of general standards that draw a baseline for cell manufacturing facilities. In recent years, stem cell researches have found great public interest in Iran and different cell therapy projects have been started in country. In this review we described the role of our tissue banking experiences in establishing a new cGMP cell manufacturing facility. The authors concluded that, tissue banks and tissue banking experts can broaden their roles from preparing tissue grafts to manufacturing cell and tissue engineered products for translational researches and phase I clinical trials. Also they can collaborate with cell processing laboratories to develop SOPs, implement quality management system, and design cGMP facilities.     

Characterization of RNA isolated from eighteen different human tissues: results from a rapid human autopsy program

Many factors affect the integrity of messenger RNA from human autopsy tissues including postmortem interval (PMI) between death and tissue preservation and the pre-mortem agonal and disease states. In this communication, we describe RNA isolation and characterization of 389 samples from 18 different tissues from elderly donors who were participants in a rapid whole-body autopsy program located in Sun City, Arizona (www.brainandbodydonationprogram.org). Most tissues were collected within a PMI of 2–6 h (median 3.15 h; N = 455), but for this study, tissue from cases with longer PMIs (1.25–29.25 h) were included. RNA quality was assessed by RNA integrity number (RIN) and total yield (ng RNA/mg tissue). RIN correlated with PMI for heart (r = ?0.531, p = 0.009) and liver (r = ?558, p = 0.0017), while RNA yield correlated with PMI for colon (r = ?485, p = 0.016) and skin (r = ?0.460, p = 0.031). RNAs with the lowest integrity were from skin and cervix where 22.7 and 31.4 % of samples respectively failed to produce intact RNA; by contrast all samples from esophagus, lymph node, jejunum, lung, stomach, submandibular gland and kidney produced RNA with measurable RINs. Expression levels in heart RNA of 4 common housekeeping normalization genes showed significant correlations of Ct values with RIN, but only one gene, glyceraldehyde-3 phosphate dehydrogenase, showed a correlation of Ct with PMI. There were no correlations between RIN values obtained for liver, adrenal, cervix, esophagus and lymph node and those obtained from corresponding brain samples. We show that high quality RNA can be produced from most human autopsy tissues, though with significant differences between tissues and donors. The RNA stability and yield did not depend solely on PMI; other undetermined factors are involved, but these do not include the age of the donor.     

Enhanced vascular biocompatibility of decellularized xeno-/allogeneic matrices in a rodent model

Glutaraldehyde preservation is the gold standard for cardiovascular biological prosthesis. However, secondary calcifications and the absence of tissue growth remain major limitations. Our study assessed in vitro and in vivo the biocompatibility of human (fascia lata, pericardium) and porcine tissues (pericardium, peritoneum) treated with a physicochemical procedure for decellularization and non-conventional pathogens inactivation. Biopsies were performed before and after treatment to assess decellularization (HE/Dapi staining/DNA quantification/MHC I/alpha gal immunostaining) and mechanical integrity. Forty-five rats received an abdominal aortic patch of native cryopreserved tissues (n = 20), treated tissues (n = 20) or glutaraldehyde-preserved bovine pericardium (GBP, control, n = 5). Grafts were explanted at 4 weeks and processed for HE/von Kossa staining and immunohistochemistries for lymphocytes (CD3)/macrophages (CD68) histomorphometry. 95% of decellularization was obtained for all tissues except for fascia lata (75%). Mechanical properties were slightly altered. In the in vivo model, a significant increase of CD3 and CD68 infiltrations was found in native and control implants in comparison with decellularized tissues (p < 0.05). Calcifications were found in 3 controls. Decellularized tissues were recolonized. GBP showed the most inflammatory response. This physicochemical treatment improves the biocompatibility of selected xeno/allogeneic tissues in comparison with their respective native cryopreserved tissues and with GBP. Incomplete decellularization is associated with a significantly higher inflammatory response. Our treatment is a promising tool in the field of tissue decellularization and tissue banking.     

Ischemic flap survival improvement by composition-selective fat grafting with novel adipose tissue derived product - stromal vascular fraction gel

Background

Flap necrosis due to insufficient blood supply is a common postoperative complication in random pattern flaps. Stem cell therapies have emerged as promising biologics for tissue ischemia. A novel fat derived product, stromal vascular fraction gel (SVF-gel), can be prepared with lipoaspirate through simple mechanical processing, removing only the lipid content. SVF-gel enriches adipose-derived stem cells and potentially beneficial for flap necrosis.

Current regulatory issues in cell and tissue therapy

Cell-based therapies have grown dramatically in power and scope in recent years. Once limited to blood and BM transplantation, these therapies now encompass tissue repair and regeneration, metabolic support, gene replacement, and immune effector functions, with established and investigational clinical applications in disorders affecting nearly every tissue and organ system. The complexity and novel applications of human cells, tissues, and cellular and tissue-based products (HCT/Ps), however, present potential risks for adverse events. The US Food and Drug Administration, responding to these concerns, has established a tiered, risk-based regulatory structure, in which more rigorous controls and safeguards are required for products thought to pose increased risk. The proposed good tissue practices (GTP) rule and existing good manufacturing practices (GMP) requirements form the principal elements of this regulatory framework. The proposed GTPs are intended to prevent HCT/P contamination with infectious disease agents, and to ensure that these cells and tissues maintain their integrity and function. GMPs focus on production of safe, pure, and potent products, and entail a higher level of process control and product characterization. All HCT/Ps will be required to comply with GTPs. HCT/Ps considered to present greater risks of adverse events, however, will be subject to both GTPs and GMPs, and must obtain premarket approval using the Investigational New Drug (IND) mechanism established for biologics. Although these requirements will present significant challenges for clinician- investigators and laboratories producing HCT/Ps, the regulations fundamentally support good clinical care by increasing safety and control, and enable good science by improving the quality and reliability of data.     

Molecular testing of <Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis> contaminating tissue allografts recovered from deceased donors

Microbiological screening of tissue allografts is crucial to prevent the transmission of bacterial and fungal infections to transplant recipients. Klebsiella was the most prevalent and resistant contaminating microorganism observed in our setting in the Iranian Tissue Bank. This study was conducted to determine the presence of extended-spectrum β-lactamase (ESBL) genes, antimicrobial resistance patterns of Klebsiella pneumoniae isolates, and their clonal relationships in allograft materials. K. pneumoniae contaminating bone and other tissue allografts recovered from deceased donors were identified and ESBL isolates were detected using a phenotypic confirmatory method. Antimicrobial susceptibility testing was carried out using the disk diffusion method. Distribution of ESBL genes and molecular typing were performed using polymerase chain reaction (PCR) and Repetitive-element (rep-PCR) methods. Of 3828 donated tissues, 51 (1.3%) were found contaminated by K. pneumoniae isolates. Compared to tissue allografts from brain-dead, heart-beating tissue donors, allografts from donors with circulatory cessation were associated with a higher risk of K. pneumoniae contamination [odds ratio (OR), 1.2 (CI 95% 0.9–2.3) (P value < 0.001)]. Half of the isolates produced ESBL, and the rate of susceptibility to cephalosporins was 51%. Among isolates, 22 (43.1%) harbored CTX-M, 31 (60.8%) SHV, and 9 (17.6%) harbored TEM types. The rep-dendrogram indicated that clones having identical or related strains with a similar antibiotype were isolated in the same period. This study provides evidence that a single clone of K. pneumoniae contaminated tissue allografts recovered from many different donors. A single clone found on tissues from several donors suggests contamination of tissues from a single source such as the tissue recovery process and environment. Genomic DNA testing and clonality of contaminating bacteria using molecular methods can focus the epidemiologic investigation on the tissue allograft recovery process including a search for contamination of the tissue recovery room environment, recovery staff, recovery equipment, reagents, solutions and supplies.     

Real-time process/quality control for HPC processing

BACKGROUND: JACIE Standards (FACT Standards in the USA) have been implemented in Europe since 1999. An on-site accreditation inspection took place at our center in January 2004. The purpose of this work was to develop a real-time process/quality control system meeting the JACIE Standards for HPC release. METHODS: Data from 194 HPC processing procedures for autologous transplantation performed over a 5-year period were analyzed. The results of different processing methods applied at our facility were compared: (1) cryopreservation without washing cells (n=50), (2) washing cells (n=87), (3) cell-density separation (n=12) and (4) positive CD34 selection (n=45). RESULTS: Four critical control points were set for the validation of HPC processing: (a) number of lost CD34(+) cells during processing, (b) contamination, (c) viability of the cells after thawing and (d) ability to reconstitute hematopoiesis after transplantation. On the basis of statistical analysis, ranges of acceptable values were defined for each critical control point and for each processing method. Those acceptable values were used for cell release and real-time quality control. DISCUSSION: This study describes a model for the validation of HPC processing and for a real-time process/quality control system for HPC release. Optimization of processing techniques, standardization of methods and comparison between facilities will open the way towards external quality controls and quality improvement.