Endoscopic versus transcranial procurement of allograft tympano-ossicular systems: a prospective double-blind randomized controlled audit

Allograft tympano-ossicular systems (ATOS) have proven their use over many decades in tympanoplasty and reconstruction after resection of cholesteatoma. The transcranial bone plug technique has been used in the past 50 years to procure en bloc ATOS (tympanic membrane with malleus, incus and stapes attached). Recently, our group reported the feasibility of the endoscopic procurement technique. The aim of this study was to assess whether clinical outcome is equivalent in ATOS acquired by using the endoscopic procurement technique compared to ATOS acquired by using the transcranial technique. A double-blind randomized controlled audit was performed in a tertiary referral center in patients that underwent allograft tympanoplasty because of chronic otitis media with and without cholesteatoma. Allograft epithelialisation was evaluated at the short-term postoperative visit by microscopic examination. Failures were reported if reperforation was observed. Fifty patients underwent allograft tympanoplasty: 34 received endoscopically procured ATOS and 16 received transcranially procured ATOS. One failed case was observed, in the endoscopic procurement group. We did not observe a statistically significant difference between the two groups in failure rate. This study demonstrates equivalence of the clinical outcome of allograft tympanoplasty using either endoscopic or transcranial procured ATOS and therefore indicates that the endoscopic technique can be considered the new standard procurement technique. Especially because the endoscopic procurement technique has several advantages compared to the former transcranial procurement technique: it avoids risk of prion transmission and it is faster while lacking any noticeable incision.     

Temporal bone bank: complying with European union directives on human tissue and cells

Background

Availability of allograft tympano-ossicular systems (ATOS) provides unique reconstructive capabilities, allowing more radical removal of middle ear pathology. To provide ATOS, the University of Antwerp Temporal Bone Bank (UATB) was established in 1988. ATOS use was stopped in many countries because of safety issues concerning human tissue transplantation. Our objective was to maintain an ATOS tissue bank complying with European Union (EU) directives on human tissues and cells.

Methods

The guidelines of the Belgian Superior Health Council, including EU directive requirements, were rigorously applied to UATB infrastructure, workflow protocols and activity. Workflow protocols were updated and an internal audit was performed to check and improve consistency with established quality systems and changing legislations. The Belgian Federal Agency of Medicines and Health Products performed an inspection to examine compliance with national legislatives and EU directives on human tissues and cells. A sample of important procedures was meticulously examined in its workflow setting next to assessment of the infrastructure and personnel.

Results

Results are reported on infrastructure, personnel, administrative workflow, procurement, preparation, processing, distribution, internal audit and inspection by the competent authority. Donors procured: 2006, 93 (45.1%); 2007, 64 (20.6%); 2008, 56 (13.1%); 2009, 79 (6.9%). The UATB was approved by the Minister of Health without critical or important shortcomings. The Ministry accords registration each time for 2?years.

Conclusions

An ATOS tissue bank complying with EU regulations on human allografts is feasible and critical to assure that the patient receives tissue, which is safe, individually checked and prepared in a suitable environment.     

Viability of cryopreserved human skin allografts: effects of transport media and cryoprotectant

Human skin allografts can be preserved by different methods. In our clinical practice, human skin allografts are harvested on multi-organ and tissue donors, transferred at +4°C in Ringer Lactate, cryopreserved with 15% Glycerol and held in the vapor phase of a liquid nitrogen freezer until delivery to the burn center. The aim of this experimental study was to evaluate the impact of transport medium and cryoprotectant on the viability of human skin allografts. For this purpose, we compared skin samples harvested from 19 multi-organ and tissue donors with two different transport media and two different cryoprotectants. Viability was assessed by the MTT assay after harvesting at laboratory reception, during storage (at +4°C) at day 2 and day 7, and after cryopreservation and thawing. Histopathological analysis was performed for each MTT assay. Results indicate that, when stored at +4°C, skin retains more viability with RPMI, whereas Glycerol and DMSO are equivalent cryoprotectants regardless of the transport medium. In conclusion, our protocol could be improved by the utilization of RPMI as transport medium.     

En bloc cervical lipectomy for treatment of the problem neck in facial rejuvenation surgery

The treatment of cervical fat in facial aesthetic surgery has received much attention in recent years. Suction lipectomy has become a very popular technique for removing cervical fat because it is easy to perform and results in few complications. This paper describes the en bloc excision of cervical fat in conjunction with rhytidectomy. The senior author has treated 1,000 patients over 17 years using this technique with a high degree of patient satisfaction and minimal morbidity. Although suction lipectomy alone may be indicated for the younger patient, our experience suggests that the en bloc excisional technique is the treatment of choice in the older patient in whom a rhytidectomy is also indicated. In contrast with suction lipectomy, we have found that the en bloc excision of cervical fat allows for more anatomic dissection and facilitates removal of greater amounts of fat and better redraping of the cervical skin.     

High frequency of in situ hybridization on thin plastic sections of human placenta with a cDNA probe for beta hCG

We describe two different techniques with plastic embedding in in situ hybridization histochemistry (ISHH). Their applicability was demonstrated by use of human placenta of the tenth gestational week and a tritium-labeled cDNA probe for the beta-subunit of hCG. In the first method, ISHH was performed on whole pieces of tissue (en bloc ISHH) pretreated with a weak acid solution, embedded in methacrylate, and sectioned at 3 microns for autoradiography. In the second technique, en bloc ISHH was carried out on tissue pre-treated with the weak acid and thereafter with detergent to further facilitate probe penetration. An acrylic resin was used for embedding, and section thickness was reduced to 1 microns. With both techniques, beta hCG cDNA/mRNA hybrids were localized exclusively to the syncytiotrophoblast (ST), in agreement with a previous study using sections of frozen placentas for hybridization to the same probe. However, owing to the higher resolution of the plastic sections the reliability of this localization was greatly increased. The number of autoradiographic grains over the acrylic resin 1-microns sections was found to be considerably higher than that over the methacrylate 3-microns sections. This study showed that treatment of tissue with detergent before en bloc ISHH, with subsequent embedding in acrylic resin and sectioning at 1 microns, gives high resolution in combination with a high signal-to-noise ratio after autoradiography. As the acrylic resin permits cutting of ultrathin sections, the results suggest that the technique may become useful for ISHH studies at the subcellular level.     

Skin allograft procurement and transplantation in Mashhad,Iran: Are burn patients’ needs being met?

Only a single donor’s tissue may save or improve lives of one hundred patients. Unfortunately, low governmental and media support of tissue procurement and transplantation programs is a worldwide problem. Loss of an effective tissue procurement program in many countries like Iran, may lead to loss of many thousands valuable tissues each year. To evaluate the rate of skin donation in Mashhad in comparison to other organs and tissues, we extracted the data related to tissue and organ procurement in Mashhad from 2001. Then we evaluated the annual skin allograft needs in the Burn Department of Imam Reza Hospital as the only referral burn center in the northeast of Iran. Brain dead potential donation rate per million populations of Mashhad in the years 2007–2014 was about 33. The mean refusal rate was 51%. Of patients who have consent for donation, more than 86% have consent for skin donation. Skin allograft procured from 119 (35.5%) candidates. Average of skin retrieval per cadaveric was 1525 cm² with a gradual increase from 1400 cm² in the first year to 1800 cm² in the last year. The recipient to donor ratio was 1.14. It is estimated that about 1 cm² of skin allograft is needed for any cm² burnt body surface area. Considering more than 700 acute burn hospitalization in our burn unit, the patients need for skin allograft would be more than 3.5 million cm², annually. The annual amount of skin procurement in Mashhad has been currently about 20,000 cm². It shows that our patients demand is higher than supply. Skin procurement and transplantation is a simple procedure which can be as lifesaving as organ procurement and transplantation. But there isn’t any national organization to regulate tissue procurement, banking and transplantation. Governmental support of skin procurement and transplantation programs especially nonprofit programs may improve skin procurement rate and save more lives of severely burnt patients.     

The Procurement Team as a Factor of Bone Allograft Contamination

We studied the effect of the procurement team on the risk of contamination in 270 large bone allografts retrieved from 53 non-living donors under strictly aseptic conditions.The overall contamination rate was 8.1%. When the procurement team was constituted by three or less members the contamination rate was 5.6%, while if there were four or more members the rate was 12.9%; this difference was significant in the statistical analysis.We conclude that a procurement team constituted by three or less trained members is a determinant factor in decreasing the bacterial contamination rate of bone allografts.     

Outcome of adrenal tissue fragments allotransplantation: The impact of cryopreservation

Cryopreservation is thought to have the potential to preserve tissue for transplantation. In addition, it can also be used for decreasing tissue immunogenicity, which might be important for prolonging allograft survival. In the present study we examined the impact of cryopreservation at various cooling rates on the outcome of allotransplantation of murine adrenal tissue fragments (ATFr). ATFr were cryopreserved with a cooling rate at 1; 10; 40 and more than 100 °C/min. After thawing it was found that the number of the cells expressing markers of dendritic cells (CD11c) and macrophages (CD11b) in the suspension obtained from ATFr decreased with increasing cooling rate. After allotransplantation the survival rates of adrenalectomized mice and the blood serum levels of corticosterone were higher in recipients of cryopreserved ATFr. By immunohistochemistry, cryopreserved allografts displayed a decreased infiltration by CD4⁺ and CD8⁺ T-lymphocytes as compared to fresh grafts. These findings suggest that cryopreserved allografts cause a less severe rejection by decreasing graft immunogenicity.     

Disinfection of human cardiac valve allografts in tissue banking: systematic review report

Cardiovascular allografts are usually disinfected using antibiotics, but protocols vary significantly between tissue banks. It is likely that different disinfection protocols will not have the same level of efficacy; they may also have varying effects on the structural integrity of the tissue, which could lead to significant differences in terms of clinical outcome in recipients. Ideally, a disinfection protocol should achieve the greatest bioburden reduction with the lowest possible impact on tissue integrity. We conducted a systematic review of methods applied to disinfect cardiovascular tissues. The use of multiple broad spectrum antibiotics in conjunction with an antifungal agent resulted in the greatest reduction in bioburden. Antibiotic incubation periods were limited to less than 24 h, and most protocols incubated tissues at 4 °C, however one study demonstrated a greater reduction of microbial load at 37 °C. None of the reviewed studies looked at the impact of these disinfection protocols on the risk of infection or any other clinical outcome in recipients.     

High-contrast en bloc staining of neuronal tissue for field emission scanning electron microscopy

Conventional heavy metal poststaining methods on thin sections lend contrast but often cause contamination. To avoid this problem, we tested several en bloc staining techniques to contrast tissue in serial sections mounted on solid substrates for examination by field emission scanning electron microscopy (FESEM). Because FESEM section imaging requires that specimens have higher contrast and greater electrical conductivity than transmission electron microscopy (TEM) samples, our technique uses osmium impregnation (OTO) to make the samples conductive while heavily staining membranes for segmentation studies. Combining this step with other classic heavy metal en bloc stains, including uranyl acetate (UA), lead aspartate, copper sulfate and lead citrate, produced clean, highly contrasted TEM and scanning electron microscopy (SEM) samples of insect, fish and mammalian nervous systems. This protocol takes 7-15 d to prepare resin-embedded tissue, cut sections and produce serial section images.     

Banking of cryopreserved arterial allografts in Europe: 20 years of operation in the European Homograft Bank (EHB) in Brussels

Vascular allografts have been used for many years in patients with infection complications and when the patient lacks own autologous venous material. Cryopreservation has permitted the long term storage of these allografts, offering the optimal solution for particular clinical situations. For more than 20 years the European Homograft Bank has prepared, stored in the liquid nitrogen vapour below ?130 °C and distributed various types of the quality controlled arterial allografts throughout the European centers and elsewhere. The tissues are prepared according to the existing European, Belgian, Swiss and other EU countries’ regulations and standards. This paper gives an overview of this activity since 1991. During this period 1,428 batches of arteries were received from recovery centres within European Union and Switzerland and 3,941 arterial segments were evaluated. 1,250 (32 %) were discarded for morphological findings (58 %), bacteriology (31 %) and other reasons, while 2,685 or 68 % (ascending and descending aorta, arch, aortic bifurcation, iliac and femoral arteries and the non-valved pulmonary bifurcations) were cryopreserved and stored. 2,506 arteries were implanted in 1,600 patients in vascular and cardiac centers in European Union and elsewhere. The most important indications were infections (65 %), critical limb ischemia (15 %) and congenital cardiac malformations (15 %). Some allografts were used for the repair of arterial injury (2 %) or prosthetic graft thrombosis (1.5 %). 10 aortic allografts (0.4 %) were used for tracheal replacement in case of cancer. In 52 cases EHB did not fulfill the surgeon’s requests due to shortage of arterial allografts. Collaboration with vascular surgeons in the tissue recovery might improve the number, diversity and quality of vascular allografts. A multicentric study is necessary to evaluate the long-term outcome of these allografts.     

Chest wall resection and reconstruction: a true thoracoscopic approach

We set out to perform resection of a chest wall mass with subsequent reconstruction using a pure thoracoscopic approach. Using video-assisted thoracic surgery via a three-incision approach, we successfully removed an 8.5 × 3.5-cm specimen en bloc. We then reconstructed the chest wall with 2-mm polytetrafluoroethylene. A total thoracoscopic approach to chest wall resection and reconstruction represent an additional option in this area of thoracic surgery. This approach avoids some of the drawbacks of more invasive procedures. This report outlines a totally thoracoscopic approach that we feel represents a safe and viable option for patients requiring chest wall resection and reconstruction.     

Analysis of predisposing factors for contamination of bone and tendon allografts

Bone and tissue allografts are widely used in transplantation. The increasing demand for safe allografts must be met, while minimizing disease transmission. We analysed the incidence and potential risk factors of allograft contamination and the effectiveness of disinfection, by reviewing 22 years of tissue bank activity and 474 donor procurements. We also compared different disinfection procedures used over the 22 years. The overall contamination rate was 10.1%. Risk factors were related to the donor or procurement method. Immediate culture at the tissue recovery site diminished the rate of false positives by reducing later sample manipulation. High-virulence allograft contamination was mainly related to donor factors, while low-virulence contamination was related to procurement methods. Analysis of donor-related risk factors showed no statistical differences for age, sex, or cause of death. An intensive care unit stay was associated with less contamination with high-virulence microbes. Procurement in a setting other than an operating theatre was associated with higher contamination rate. Team experience reduced contamination. Pelvic and tendon allografts were most frequently contaminated. Proper disinfection considerably reduced the contamination rate to 3.6%. We conclude that procurement must be performed under aseptic conditions, with short delays, and by trained personnel. Grafts should be disinfected and packed as soon as possible.     

Assessment of cryopreserved donor skin viability: the experience of the regional tissue bank of Siena

Skin allografts from cadaver donors are an important resource for treating extensive burns, slow-healing wounds and chronic ulcers. A high level of cell viability of cryopreserved allografts is often required, especially in burn surgery, in Italy. Thus, we aimed to determine which conditions enable procurement of highly viable skin in our Regional Skin Bank of Siena. For this purpose, we assessed cell viability of cryopreserved skin allografts procured between 2011 and 2013 from 127 consecutive skin donors, before and after freezing (at day 15, 180, and 365). For each skin donor, we collected data concerning clinical history (age, sex, smoking, phototype, dyslipidemia, diabetes, cause of death), donation process (multi-tissue or multi-organ) and timing of skin procurement (assessment of intervals such as death-harvesting, harvesting-banking, death-banking). All these variables were analysed in the whole case study (127 donors) and in different groups (e.g. multi-organ donors, non refrigerated multi-tissue donors, refrigerated multi-tissue donors) for correlations with cell viability. Our results indicated that cryopreserved skin allografts with higher cell viability were obtained from female, non smoker, heartbeating donors died of cerebral haemorrhage, and were harvested within 2 h of aortic clamping and banked within 12 h of harvesting (13–14 h from clamping). Age, cause of death and dyslipidaemia or diabetes did not appear to influence cell viability. To maintain acceptable cell viability, our skin bank needs to reduce the time interval between harvesting and banking, especially for refrigerated donors.     

Effect of freezing and storage temperature on stability and antimicrobial activity of an antibiotic mixture used for decontamination of tissue allografts

One of the most important risks to be controlled in tissue banking is the infection associated with the clinical use of auto- and allografts. Thus, tissue disinfection protocols are used, in addition to processing in controlled environments. For this purpose, combinations of antibiotics are designed to ensure a broad spectrum of antimicrobial activity. This type of protocol is usually validated by testing its antimicrobial efficacy. In this work, we have studied the effect of several factors on the potential of an antibiotic mixture: container, freezing, storage at 4 °C, storage at ??30 °C and storage at ??80 °C. The molecular stability of the compounds has also been tested, additionally to their efficacy. Our findings show that storage conditions affect the molecular stability of Fungizone and Tobramycin (only in case of frozen storage for the last one). Nevertheless, the solution retains its antimicrobial activity for several weeks. The availability of stored aliquots of disinfectant solution and defining expiry dates for different storage conditions can help to schedule tissue bank tasks.     

Cartilage storage at 4 °C with regular culture medium replacement benefits chondrocyte viability of osteochondral grafts in vitro

Maintenance of articular cartilage allografts in culture media is a common method of tissue storage; however, the technical parameters of graft storage remain controversial. In this study, we examined the optimal temperature and culture medium exchange rate for the storage of osteochondral allografts in vitro. Cylindrical osteochondral grafts (n = 120), harvested from the talar joint surface of ten Boer goats, were randomly classified into four groups and stored under the following conditions: Group A1 was maintained at 4 °C in culture medium that was refreshed every 2 days; Group A2 was maintained at 4 °C in the same culture medium, without refreshing; Group B1, was maintained at 37 °C in culture medium that was refreshed every 2 days; Group B2, was maintained at 37 °C in the same culture medium, without refreshing. Chondrocyte viability in the grafts was determined by ethidium bromide/fluorescein diacetate staining on days 7, 21, and 35. Proteoglycan content was measured by Safranin-O staining. Group A1 exhibited the highest chondrocyte survival rates of 90.88 %, 88.31 % and 78.69 % on days 7, 21, and 35, respectively. Safranin O staining revealed no significant differences between groups on days 21 and 35. These results suggest that storage of osteochondral grafts at 4 °C with regular culture medium replacement should be highly suitable for clinical application.     

Assessment of organ culture for the conservation of human skin allografts

Objective Human skin allografts are used in the treatment of severe burns and their preservation is therefore critical for optimal clinical benefit. Current preservation methods, such as 4°C storage or cryopreservation, cannot prevent the decrease of tissue viability. The aim of this study was to assess viability and function of skin allografts in a new skin organ culture model, allowing conservation parameters as close as possible to physiological conditions: 32°C, air–liquid interface and physiological skin tension. Design Twelve skin samples, harvested from 6 living surgical donors, were conserved 35 days in two conditions: conservation at 4°C and organ culture. Viability and function of skin samples were investigated at Day 0, 7, 14, 21, 28 and 35 using cell culture methods (trypan blue exclusion, Colony Forming Efficiency and Growth Rate), histopathological and histoenzymological studies (Ki67 immunostaining). Results In the two conditions, fibroblast and keratinocyte viability was progressively affected by storage, with a significant decrease observed after 35 days. No statistical difference could be observed between the two conditions. The two methods were also comparable regarding alterations of fibroblast and keratinocyte culture parameters, which were respectively significantly reduced at Day 7 and 21, compared to fresh skin. By contrast, histopathological and histoenzymological studies revealed a better preservation of skin architecture and proliferative potential at 4°C, as compared to organ culture. Conclusion These results indicate that skin organ culture does not provide significant advantages for skin allograft preservation. However, its potential use as an experimental model to study skin physiology and wound healing should be further evaluated.     

Activity of four antimicrobial cocktails for tissue allograft decontamination against bacteria and Candida spp. of known susceptibility at different temperatures

Several antimicrobial cocktail solutions of differing composition and concentrations are widely used to decontaminate viable banked tissue allografts at different temperatures and times of exposure. We compared the efficiency of four cocktails comprising nine antimicrobials to kill suspensions of a panel of 27 strains of 13 bacterial species, and 3 Candida spp. at 4, 22 and 37 °C for 24 h. All but one bacterial strains were susceptible to one or more of the agents tested individually at concentrations at least fourfold below the recommended susceptibility breakpoint minimum inhibitory concentrations for drug/species combinations. Candida lusitaniae was resistant to nystatin and amphotericin. The concentrations of several of the cocktail constituents were often greatly in excess (50–1,000-fold) of that required to inhibit the growth of susceptible strains. All cocktails were ineffective against a pan-resistant strain of Enterococcus faecium and one of the four cocktails failed to kill two strains of methicillin resistant Staphylococcus aureus. Each cocktail was most efficient at 37 °C, less so at 22 °C, and poorly active at 4 °C. We conclude that the practice of decontamination of tissues with antimicrobials at low temperatures is not supported by in vitro susceptibility tests.     

Broth versus solid agar culture of swab samples of cadaveric allograft musculoskeletal tissue

As part of the donor assessment protocol, bioburden assessment must be performed on allograft musculoskeletal tissue samples collected at the time of tissue retrieval. Swab samples of musculoskeletal tissue allografts from cadaveric donors are received at the microbiology department of the South Eastern Area Laboratory Services (Australia) to determine the presence of bacteria and fungi. This study will review the isolation rate of organisms from solid agar and broth culture of swab samples of cadaveric allograft musculoskeletal tissue over a 6-year period, 2006–2011. Swabs were inoculated onto horse blood agar (anaerobic, 35 °C) and chocolate agar (CO₂, 35 °C) and then placed into a cooked meat broth (aerobic, 35 °C). A total of 1,912 swabs from 389 donors were received during the study period. 557 (29.1 %) swabs were culture positive with the isolation of 713 organisms, 249 (34.9 %) from solid agar culture and an additional 464 (65.1 %) from broth culture only. This study has shown that the broth culture of cadaveric allograft musculoskeletal swab samples recovered a greater amount of organisms than solid agar culture. Isolates such as Clostridium species and Staphylococcus aureus would not have been isolated from solid agar culture alone. Broth culture is an essential part of the bioburden assessment protocol of swab samples of cadaveric allograft musculoskeletal tissue in this laboratory.