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.     

The effectiveness of bone banking in Central Serbia: audit of the first seven years

We analyzed the incidence and predisposing factors for overall discard rate after retrieval of 295 femoral head allografts. The aim of the present study was to evaluate the quality system of institutional bone banking and to ensure that we can provide high standard allografts with low infection rate. Audit of bone banking was conducted on 295 donors and 180 recipients. Of the 295 donated femoral heads 77 were discarded, giving an overall discard rate of 26.1 %. At retrieval, 37 allografts were positive, giving an overall contamination rate of 12.54 %. The organism most commonly identified was Staphylococcus species. Seven (2.37 %) of the 295 allografts failed the blood screening tests. Twelve allografts (4.06 %) were discarded because of suspected damage of the packaging or disuse during surgery. Due to donor death or inability to perform serology retests, 21 (7.11 %) allografts were discarded. In the postoperative survey an infection rate of 2.22 % was found. After 7 years of bone banking, our results show that overall discard rate and allograft related infection rate are in accordance with the international standards. The leading cause of allograft discarding was bacterial contamination influenced by the surgical team. We suggest stringent aseptic allograft handling during harvesting and thawing within highly concentrated antibiotic solution to reduce a possibility of its contamination.     

Validation of 15 kGy as a radiation sterilisation dose for bone allografts manufactured at the Queensland Bone Bank: application of the VD<Subscript>max</Subscript> 15 method

Background ISO 11137-2006 (ISO 11137-2a 2006) provides a VD_max 15 method for substantiation of 15 kGy as radiation sterilisation dose (RSD) for health care products with a relatively low sample requirement. Moreover, the method is also valid for products in which the bioburden level is less than or equal to 1.5. In the literature, the bioburden level of processed bone allografts is extremely low. Similarly, the Queensland Bone Bank (QBB) usually recovers no viable organisms from processed bone allografts. Because bone allografts are treated as a type of health care product, the aim of this research was to substantiate 15 kGy as a RSD for frozen bone allografts at the QBB using method VD_max 15—ISO 11137-2: 2006 (ISO 11137-2e, Procedure for method VD_max 15 for multiple production batches. Sterilisation of health care products – radiation – part 2: establishing the sterilisation dose, 2006; ISO 11137-2f, Procedure for method VD_max 15 for a single production batch. Sterilisation of health care products – radiation – part 2: establishing the sterilisation dose, 2006). Materials 30 femoral heads, 40 milled bone allografts and 40 structural bone allografts manufactured according to QBB standard operating procedures were used. Method Estimated bioburdens for each bone allograft group were used to calculate the verification doses. Next, 10 samples per group were irradiated at the verification dose, sterility was tested and the number of positive tests of sterility recorded. If the number of positive samples was no more than 1, from the 10 tests carried out in each group, the verification was accepted and 15 kGy was substantiated as RSD for those bone allografts. Results The bioburdens in all three groups were 0, and therefore the verification doses were 0 kGy. Sterility tests of femoral heads and milled bones were all negative (no contamination), and there was one positive test of sterility in the structural bone allograft. Accordingly, the verification was accepted. Conclusion Using the ISO validated protocol, VD_max 15, 15 kGy was substantiated as RSD for frozen bone allografts manufactured at the QBB.     

Analysis of potential contamination factors in musculoskeletal tissues

Tissue Banks have become the main source for bone grafts, due to preference for homologous tissues. Notwithstanding the use of aseptic techniques for procurement of tissues and judicious selection of donors, microorganisms are frequently found in procured bones. Purpose of this study is to evaluate the factors that increase safety of procurement and minimize discard of procured tissues. Microbiological contamination was analyzed in 1271 musculoskeletal tissues removed from 138 multi-organ donors over a period extending from 2006 to 2016. Effects of various risk factors related with contamination were estimated using a logistic regression model. Microbiological contamination rate in the tissues was 17.1%; low pathogenic microorganisms were cultivated in 12.9% of the tissues, while highly pathogenic ones were cultivated in 4.2% of the tissues. Evolution of one single team was monitored during that period, verifying a fall in the general contamination level from 22.5 to 9.2%. Absence of antibiotics increased low pathogenic contamination risk. Every additional day in intensive care unit (ICU) increased the risk of highly pathogenic contamination. Time elapsed between death and the beginning of removal procedures was found to be relevant for both low pathogenic and highly pathogenic microorganisms. Among the studied factors, the following contributed for a significant increase in contamination by microorganisms in removed tissues: lack of use of prophylactic antibiotic therapy in donors, quantity of removed tissues, length of admission in ICU and the time elapsed between aortic clamping and beginning of the removal procedure.     

Very long-term radiographic and bone scan results of frozen autograft and allograft bone grafting in 17 patients (20 grafts) a 30- to 35-year follow-up

In the early 1950s, 48 patients received bone implants from a bone bank in Tel-Hashomer Hospital that stored frozen autograft and allograft bones at temperatures less than -17 degrees C. Seventeen (35%) of these patients (20 implants), 10 men and 7 women, with a mean age of 52.4 (34-69) years were available for follow-up after a mean period of 32.5 (30-35) years. They underwent clinical examination, radiographs and bone scans to evaluate their surgical results. Fracture healing, non-union, graft resorption, osteoporosis and bone sclerosis were used as radiographic criteria for bone incorporation, and normal, increased and decreased uptake served to assess the bone scan. Based on the above criteria, the results were satisfactory in 17 (85%) and poor in 3 (15%). The three failures were after shelf operation for hip dysplasia that used two allografts and one autograft. Allogenous or a combination of allogenous with autogenous frozen bone grafts proved to be a satisfactory and durable method for filling bone defects.     

Development of a bacteriophage model system to investigate virus inactivation methods used in the treatment of bone allografts

Bone allografts are commonly used in a variety of surgical procedures, to reconstruct lost bone stock and to provide mechanical support during the healing process. Due to concerns regarding the possibility of disease transmission from donor to recipient, and of contamination of grafts during retrieval and processing procedures, it is common practice to sterilise bone allografts prior to issue for clinical use. It is vital that the sterilisation processes applied to allografts are validated to demonstrate that they achieve the required level of bioburden reduction, and by extension that validated models are used for these studies. Two common sterilisation protocols applied to bone allografts are gamma irradiation and ethylene oxide gas sterilisation, and there are currently no validated models available for measuring the anti-viral efficacy of ethylene oxide treatment with regard to bone allografts or readily useable models for assessing the anti-viral efficiency of gamma irradiation treatment. We have developed and validated models for both these sterilisation processes, using the bacteriophage ϕ×174, and utilised the models to measure the antiviral activity of the standard ethylene oxide and gamma irradiation sterilisation processes applied to bone allografts by the National Blood Service. For the irradiation model, we also utilised bacterial spores (Bacillus pumilus). Our results show that ethylene oxide sterilisation (which can only be applied to lyophilised grafts) inactivated >6.1log₁₀ of the model virus, and gamma irradiation (at 25–40 kGy and applied to frozen allografts) inactivated 3.6–4.0log₁₀ of the model virus and >4log₁₀ of the bacterial spores. Gamma irradiation at this dosage is therefore not in itself a sterilisation process with respect to viruses.     

Effects of gamma irradiation on mechanical properties of defatted trabecular bone allografts assessed by speed-of-sound measurement

New sterilization methods for human bone allografts may lead to alterations in bone mechanical properties, which strongly influence short- and medium-term outcomes. In many sterilization procedures, bone allografts are subjected to gamma irradiation, usually with 25 KGy, after treatment and packaging. We used speed-of-sound (SOS) measurements to evaluate the effects of gamma irradiation on bone. All bone specimens were subjected to the same microbial inactivation procedure. They were then separated into three groups, of which one was treated and not irradiated and two were exposed to 10 and 25 KGy of gamma radiation, respectively. SOS was measured using high- and low-frequency ultrasound beams in each orthogonal direction. SOS and Young modulus were altered significantly in the three groups, compared to native untreated bone. Exposure to 10 or 25 KGy had no noticeable effect on the study variables. The impact of irradiation was small compared to the effects of physical or chemical defatting. Reducing the radiation dose used in everyday practice failed to improve graft mechanical properties in this study.     

Comparison of frozen and freeze-dried particulate bone allografts

Freeze-dried and frozen particulate bone allografts are used interchangeably on the assumption that the biologic behavior of these grafts is similar. Dissimilarities in biologic behavior and differences in the rate and extent of bone incorporation of freeze-dried and frozen particulate grafts were demonstrated in a comparative study using a non-human primate model. Freeze-dried particulate allografts induced new bone formation and healing of the osseous defects much faster than the frozen allografts.     

Endoscopic procurement of tympano-ossicular allografts: alternative to the transcranial or retroauricular technique

A tympano-ossicular tissue bank complying with European Union 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. The transcranial procurement technique has become the standard approach to procure tympano-ossicular allografts since the 1970s because it can provide en bloc allografts. Over the last 10–20 years, en bloc allografts have been abandoned and only the malleus (hammer) is left attached to the tympanic membrane. This modification enables introduction of the transmeatal procurement technique. Transmeatal procurement using readily available nasal 0° and 30° endoscopes is a feasible alternative which avoids contact with the dura mater and is not esthetically invasive to the donor. It involves a more time-consuming procurement but avoids the need for preparation of the temporal bone plug and is therefore generally more time-efficient.     

Tissue banking in Mexico

Introduction: Here, we describe our Tissue Banking experiences of 4 years of activity in Mexico. Methods: Data of allografts provided by our Bank and bone retrievals performed by our teams between February of 2001 and August of 2004 were included. Results: There were 100 bone donors, a total of 1107 tissues were obtained with an average of 11 tissues by retrieval, samples from all tissues were obtained during retrieval and cultured for bacterial contamination, 250 tissues were positives to bacterial growth with an average of 22.58% of bacterial contamination of tissue by retrieval. A total of 4493 allografts were provided and were utilized in 3643 patients. The allografts were used mainly by orthopedic surgeons (62%) and dentists (30%). The most used allografts were morcellized cancellous bone 31%, pulverized 25% and chips of cancellous bone 20%. Among orthopedic patients the most frequent procedures were related with spine degenerative diseases 39.09%, followed by non-pathological fractures and its complications 28.67% and bone tumors and cystic bone lesions 11.59%. Conclusions: Sustained increase of allograft utilization in Mexico reflects a great necessity for them in our country. The increase in public awareness about tissue donation has allowed an increase in tissue donations and retrievals.     

Contamination of tissue allografts from a deceased donor through haematic dissemination: a case study

Infection is one of the most dangerous complications that can be seen when implanting bone or tendon allografts from a deceased donor. The most common germs isolated are found among the cutaneous florae, but sometimes they may be present in the bloodstream as a result of severe injuries suffered before the time of the decease. We present a case of contamination of allografts in a musculoskeletal tissue donor deceased after an accident, whose allografts were contaminated by gastrointestinal microorganisms, probably disseminated through the donor’s blood.     

Bone bank service in Odense, Denmark. Audit of the first ten years with bone banking at the Department of Orthopaedics, Odense University Hospital

There has been an increase in the demand for allograft bone in recentyears. The Odense University Hospital bone bank has been in function since1990,and this paper outlines our results during the 10 year period 1990–1999.Potential donors were screened by contemporary banking techniques which includea social history, donor serum tests for HIV, hepatitis B and C, and graftmicrobiology. The bones were stored at –80 °C. No typeofsecondary sterilisation was made. 423 femoral heads were approved and donatedto300 patients,1–6 heads/operation. The allografts have been used mainly toreconstruct defects at revision hip arthroplasty (34%), and for fracturesurgery(24%). 7 % of all transplanted patients were reoperated because of infection.Inthe hip revision group the infection rate was 4 %. There were no cases ofdisease transmission. During the 10 year period there was a change in theclinical use of the allografts. In the first years the allografts were mainlyused for spinal fusion surgery, but today the majority are used in hip revisionand fracture surgery. The clinical results correspond to those reported inlarger international series.     

Effects of Hydrogen Peroxide Cleaning Procedures on Bone Graft Osteoinductivity and Mechanical Properties

Bone allografts are frequently used during orthopaedic trauma cases or other reconstructive procedures. Most allografts are processed and cleaned before use. Our goals were to determine if an improved cleaning procedure compromises the strength or osteoinductivity of a graft. We compared our improved cleaning procedure to our standard cleaning procedure on cortical bone allograft. The cleaning procedures are generally composed of a series of chemical steps with nonionic detergents, hydrogen peroxide, and alcohol under time and temperature control, subjected to ultrasonic agitation. We tested the compressive strength, impact strength, and shear strength following the standard and improved cleaning procedures. Osteoinductivity was tested in 4 groups, using the improved cleaning procedure with four different hydrogen peroxide cleaning times: 0, 1, 3, and 5 h. Osteoinductivity was evaluated in vivo, using a 28-day implant in the hamstring muscle of an athymic, nude mouse. Results demonstrated that osteoinductivity is maintained with cleaning in hydrogen peroxide for up to 1 h, and that compressive strength, impact strength, and shear strength were all unaffected by the improved cleaning procedure. The improved cleaning procedure therefore did not compromise the strength or osteoinductivity of cortical bone allografts in comparison to the standard procedure.     

Comparison of contamination of femoral heads and pre-processed bone chips during hip revision arthroplasty

With bone impaction grafting, cancellous bone chips made from allograft femoral heads are impacted in a bone defect, which introduces an additional source of infection. The potential benefit of the use of pre-processed bone chips was investigated by comparing the bacterial contamination of bone chips prepared intraoperatively with the bacterial contamination of pre-processed bone chips at different stages in the surgical procedure. To investigate baseline contamination of the bone grafts, specimens were collected during 88 procedures before actual use or preparation of the bone chips: in 44 procedures intraoperatively prepared chips were used (Group A) and in the other 44 procedures pre-processed bone chips were used (Group B). In 64 of these procedures (32 using locally prepared bone chips and 32 using pre-processed bone chips) specimens were also collected later in the procedure to investigate contamination after use and preparation of the bone chips. In total, 8 procedures had one or more positive specimen(s) (12.5 %). Contamination rates were not significantly different between bone chips prepared at the operating theatre and pre-processed bone chips. In conclusion, there was no difference in bacterial contamination between bone chips prepared from whole femoral heads in the operating room and pre-processed bone chips, and therefore, both types of bone allografts are comparable with respect to risk of infection.     

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.     

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.     

Processing of cardiovascular allografts: effectiveness of European Homograft Bank (EHB) antimicrobial treatment (cool decontamination protocol with low concentration of antibiotics)

To assess the effectiveness of antimicrobial treatment by using cool decontamination protocol with low concentration of antibiotics during processing of cardiovascular allografts, 948 allografts processed during a 2-year period were analysed. Five hundred and fourty one donors aged <62 years were classified in: multiorgan donors (MOD) with non-transplantable hearts; recipients of cardiac transplantation (RHT); and non-beating heart cadavers with a warm ischemic time of less than 6 h (NBHD). During processing three samples for bacteriology testing were taken A (sampling before decontamination); B (sampling after decontamination); C (sampling on the final product). Samples A were positive in 348 cases (36.4%), respectively 36% for MOD, 21.6% for RHT and 78.1% for NBHD. All the allografts were immersed in a cocktail of four antibiotics at 4 °C. After exposure to antibiotics the rate of decontamination of those with A positive was 90.4, 92.5, 82.5% respectively for MOD, RHT, NBHD. At the end of processing, 57 allografts (6%) were positive in B and/or C, 15 allografts remained contaminated with the same bacteria as in A, 42 were contaminated during processing. The overall rate of sterility in the end of processing is 94% and for each group this is: 95.4% for MOD, 96.8% for RHT and 86.3% for NBHD. Analysis shows that there is no influence of time of exposure in AB in the rate of decontamination for MOD and RHT. The most predominant germ in contamination is Coagulase Negative Staphylococcus (CNS) (53.4% alone, 8.9% with other bacteria). 83.3% of MOD; 88.5% of RHT were contaminated with one germ, while 40.4% of NBHD were contaminated with more than one. This revised version was published online in July 2006 with corrections to the Cover Date.     

The Analysis of Bacterial Results in the Kidney Perfusion Fluid and Bone Allografts in Human Multiorgan Donors

In 19 young human multiorgan donors, we simultaneously analyzed the bacterial contamination of the kidney perfusion fluid and all retrieved bone allografts. Donor exclusion criteria were done according to the American and European Association of Tissue Banks excluding all patients with perforating wounds. The kidney perfusate revealed a contamination in 17 of 19 (89.5%) donors. Allograft testing demonstrated positive bacterial growth in 34 of 76 allografts (44.7%). Microorganisms originated from the normal skin flora and could be related to contamination during the harvesting procedure. In 5 cases we cultured identical bacterial subspecies in both cultures as a possible sign for systemic bacterial spreading during the multiorgan harvesting procedure.     

Limitations of Bone Harvesting from Organ Donors in Poland

The demand for organ transplantations greatly exceeds the number of organ donors (OD) and it is likely that this discrepancy will continue. There is a continuously growing demand for biostatic allogenic bone transplantation mostly for orthopedic reconstructions. The bone material is predominantly harvested during postmortem examinations in forensic medicine departments. The collected amounts are not enough so the material need to be acquired from other sources. Bone collecting from OD seems to be the optimal solution. Between January 2000 and December 2005, 1,883 out of 2,601 organ donors fulfilled the criteria for bone donation, but only 42 bone harvesting (BH) were performed being only 2.22% of all suitable cases. The main reasons for so few bone procurements were: lack of acceptance of bone procurement by relevant part of donors’ families, insufficient cooperation between tissue banks and transplantation teams, lack of equipment for BH, and no experience in bone procurement. This paper was presented and discussed at the 14th International Congress of the European Association of Tissue Banks in Florence, Italy (8–11 December, 2005).     

Analysis of potential factors affecting allografts contamination at retrieval

The microbiological contamination of retrieved tissues has become a very important topic and it is a critical aspect in the safety of allografts, especially from multi-tissue donors whose tissues are frequently contaminated as a consequence of retrieval. We analysed a total of 10,107 tissues, 8178 musculoskeletal and 1929 cardiovascular tissues, retrieved from 978 multi-tissue donors. Of these, 159 heart-beating donors (HBD) were also organ donors, while the remaining 819 non-heart-beating donors (NHBD) were tissue donors only. A multivariate logistic model was used to determine the factors affecting contamination risk during retrieval. In the model, the dependent variable was the presence/absence of contamination while the covariates included were: gender, type of donor, age of donor, cause of death, previous skin donation, cadaver time, number of people attending the retrieval, number of tissues retrieved. Moreover, a second log-linear model was used to determine the number of strains isolated per tissue. Tissue contamination was statistically correlated with gender, type of donor, cadaver time, number of people attending the retrieval and season. In conclusion, to minimize the risk of bacterial contamination, aseptic techniques should be used at retrieval, with the number of retrieval team members kept to a minimum. In addition, cadaver time should be as short as possible and the donor should be refrigerated within a few hours after death.