Microbiological monitoring of bone grafts: two years' experience at a tissue bank

Journal of Hospital Infection (1998) 38, 261-271

Microbiological monitoring of bone grafts: two years’ experience at a tissue bank

M. Farrington, I. Matthews, J. Foreman*, K. M. Richardson* and E. Caffrey”

Clinical Microbiology and Public Health Laboratory, Addenbrooke’s Hospital, Cambridge CB2 2QW, and “East Anglia Tissue Bank, East Anglia Blood

Centre, Long Road, Cambridge CR2 2PT UK

Received 3 July 1997; revised manuscript accepted 22 October 1997

Summary: In the first two years of operation of a tissue bank, bone was processed on 63 occasions from 22 cadaveric donors and on 37 occasions from 1 X5 living donors. A standardized protocol for microbiological sampling, culturing and interpretation of the results was developed. Semi-quantitative culture of washings of bone was performed on receipt by the tissue bank, and broth enrichment cultures of bone samples were performed at the end of processing, and again after irradiation. One bone donation was rejected because of heavy contamination with Klebsiella sp. on receipt, and contamination of six donations with Burkholderia cepacia was shown to have come from a water deionizer. Contamination of bone on receipt by the tissue bank decreased during the study period, probably related to increasing experience of staff harvesting bone. Microbiological surveillance of bone grafts protect recipients from infection, and is useful as a quality control of the process of bone banking.

Keywords: Bone bank; allograft bone; bacterial contamination; microbiological surveillance; quality control.

Introduction

Although general standards for the performance of tissue banking in the UK have been established,’ no detailed guidelines for microbiological surveillance of bone banking are available. Thus, bone banks and their local collaborating microbiology laboratories have no consistent guidance on the appropriate stages of procurement and processing for sampling to be performed, on sampling methods, on culture media and incubation conditions and times, or on the interpretation of results. Several North American bone banks have developed monitoring procedures,*” and the American Association of Tissue Banks’ has published detailed guidance on sampling, laboratory methods and interpretation. However, transatlantic experience with fresh bone transplants is not directly applicable to the UK,

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262 M. Farrington et al.

where much processed bone is terminally sterilized by irradiation or ethylene oxide exposure before transplantation. One British group has recently published their experience with bacteriological monitoring of allografts from living donors,’ but their procedures involved only storage of grafts in a bone bank, with processing and fashioning of bone performed in the operating theatre at the time of implantation. Not surprisingly, therefore, there is debate in the UK over the need and methods for bacteriological monitoring of bone for transplant and over the interpretation of positive results, and there is wide variation in practice among bone banks.

We have developed local procedures, and we wish to report our experience of their use during the first two years of the establishment of a bone bank.

Methods

Processing of tissue in the East Anglia Tissue Bank is carried out in a suite of ‘class 100’ clean rooms, designed to minimize the risks of external contamination. In-house standard operating procedures for cleaning and microbiological monitoring are in place to achieve the requirements for environmental hygiene.

Potential donors are screened for suitability of their tissues for surgical use and for freedom from risks of transmitting infections to the recipient.’ Femoral heads are recovered during primary hip replacement by appropriate surgical techniques. Other musculoskeletal tissues are retrieved in the mortuary, where all reasonable measures are taken to reduce microbial contamination by extensive use of antiseptics and no-touch techniques, although it is not possible to use full aseptic procedure.

Figure 1 illustrates the flow of processing and sampling procedures used for morcellized and fashioned bone. Femoral heads from living donors were batched in groups of five. Each piece or batch of bone entering the bank was washed in 500 mL 0.9% sodium chloride solution. This solution was then returned to its supply bag, the line clipped, and sent rapidly to the microbiology laboratory. Two hundred microlitre samples of wash solution were inoculated to three blood agar plates which were incubated for three days at 37°C in air with 10% added CO*, for three days anaerobically, and for five days in air at room temperature. A selective Sabouraud’s agar plate was also inoculated with 200 FL wash solution and incubated for five days in air at 25°C. A growth of 1Ocfu per plate was, therefore, equivalent to 50 cfu/mL organisms in the original wash solution. Staphylococcus aweus, P-haemolytic streptococci, Clostridium spp., Enterobacteriaceae and pseudomonads comprised a list of ‘specific pathogens’ indicating poor quality. Growth of specific pathogens at >50 cfu/mL of wash solution, or other growth at >lOO cfu/mL, was taken as a ‘significantly’ positive result, and such bone was not transplanted.

We assumed that tissues entering the bank were contaminated with bacteria. Surface decontamination of bone before processing in the aseptic

Bone graft monitoring 263

Harvested bone received in tissue bank

Hold pending serological and culture results

Hold pending serological and culture results

I:igure 1. 1~10~ of processing and sampling procedures in the EA Tissue Bank. lL1, bone morcellized during processing; F, hone fashioned during processing; p, samples sent in universal containers to microbiology laboratory: p>\, wash fluid sample; up, paired processing samples; p,, paired irradiation samples. ‘k, Bone graft plus irradiation samples sent for irradiation (25 kGy).

suite was carried out in a separate area to reduce the risk of introducing micro-organisms to the clean rooms. Bone was received in the bank sealed within plastic bags which had a sterile luer connector attached to them. Five hundred millilitres of a 0.1%) solution of peracetic acid in 25% ethanol/ water was placed in a sterile aspirator which was attached to a sterile silicone rubber tube with an attached luer connector. About 400 mL of this solution was then run in to the bag containing the bone, while the aspirator was kept at the same level as the bag of bone so the remaining solution acted as a trap to prevent entry of air. For 10 min the bag was rocked on an orbital shaker. The solution was allowed to run back in to the aspirator under gravity, and the luer connector was sealed with a blank adaptor. The bag containing the bone was then transferred to the bone processing room in the aseptic suite.

After fashioning or morcellising the bone, before it was sealed in im- permeable plastic bags, two sets of duplicate samples of bone (each ap- proximately 1 g in weight) were collected in to sterile universal containers. One pair of these samples was sent for culture at this stage, whereas the other pair accompanied the processed tissue for gamma irradiation (25-30 KGy) and were sent for culture upon their return. These ‘processing’ and ‘irradiation’ samples were incubated at 37°C in air for two days after addition of brain-heart broth on the open bench in the microbiology


Table I. SigniJicant positive cultures

Donor Number Processing Positive samples type sessions

Wash Processing Irradiation

Cadaveric 22 63 1 (Klebsiella sp.) (6 :NS.

1 CNS’ (GIGS)

+ B cepacia) Living 185 37 0

(2 B. Lpacia; 0

1 CNS)

CNS, wag&se-negative staphylococcus.

laboratory, and then 10 PL samples of broth were subcultured to blood agar plates incubated aerobically and anaerobically as above, and a 100 PL sample was inoculated to a Sabouraud agar plate. Irradiation cultures were not performed on nine early bone specimens from living donors, before procedures were finalized. Growth of specific pathogens from both duplicate samples of the processing or irradiation sample of bone was an indication for further investigation and possible rejection, but isolation of other organisms from the processing sample was regarded as acceptable for transplantation provided that irradiation cultures turned out to be sterile. A positive result from only one of the duplicate samples was considered to represent culture contamination. Results were collated over time and as- sessed monthly by a consultant microbiologist who looked for recurrent but intermittent isolates that might represent a common source of low- level contamination. Organisms isolated in significant concentrations from single and duplicate samples from all three monitoring stages were stored in glycerol broth at -70°C to enable typing and investigation of possible common sources.

After return from irradiation, bone grafts were stored at -80°C and not used until the results of culture and pre-processing screens were available;’ in the East Anglia Tissue Bank bone is stored at -80°C for up to five years. Each bone package released for grafting was accompanied by a form on which the surgeon was asked to report any early post-operative problems of engraftment or infection.

Results

Between 9 February 1993 and 14 December 1994, bone was processed on 63 occasions from 22 cadeveric donors, and on 37 occasions from 185 living donors. Table I summarizes the ‘significant’ results obtained. Klebsiella sp. was isolated in pure, confluent growth from one wash solution from a cadaveric donation, and this bone was not transplanted, although the

Bone graft monitoring

Table I I. Total positive cultures j~om cadaaeric and lioing donors

265

Organism

Cadavcric donors CNS Klehsiella snn.

. &

Acinetohacter “pp. B. cepacia Bacillus spp. Ps. aeruginosa Staph. aureus Penicilliuvn sp. CNS + diphtheroid Other mixtures*

Total I,iving donors

CNS B. cepacia

Total

Wash solution Processing sample Irradiation sample

10 26 2 2 1

1 2 4

I 1

4 1

21 (3&l) 37 (2LX) 8 (6$3)

7 1

14 (lL%l) 1 (3%‘%,)

‘I’hcrc were 63 wash solutions and 126 processing and irradiation samples from cadaveric donors, and 37 wash solutions, 74 processing samples and 56 irradiation samples from batches of boric from living donors (irradiation samples were not cultured in nine early donations). All positive cultures are included, those classitied as ‘significant’ as well as ‘not significant’. *Includes <,ne (‘andida sp.

subsequent samples were sterile. All other bone was released for transplantation. Coagulase-negative staphylococci (CNS) were isolated from both duplicate samples of the irradiation specimen from one cadaveric donation, but the corresponding wash solution and processing sample cultures were sterile, and two packets of ground bone from the donation were submitted for broth culture and no organisms were recovered.

Table II shows all the organisms grown from cultures from cadaveric and living donations, including those classified as ‘significant’ and ‘contaminants’. Organisms were isolated from 21 (33,3%) of the 63 single samples of wash solution from cadaveric donors, 37 (29.4%) of 126 duplicate samples of processed bone, and eight (6.3%) of 126 duplicate samples of bone after irradiation. No bacteria were isolated from any wash solution in the living donor group. Isolates from processing samples from living donors were equally divided between CNS and a pseudomonad related to Burkholderia (formerly known as Pseudomonas) cepacia, identified by Dr B Holmes of the National Collection of Type Cultures, Central Public Health Laboratory, Colindale. CNS were the commonest isolates overall, and the majority of other isolates were also members of the normal human skin flora.

Bone from six donations from two cadaveric and four living donor batches over a four-week period was found to contain B. cepacia. All isolates came only from processing samples (three in only one duplicate, and three in both duplicates), and all of the corresponding wash and post-irradiation


Figure 2. Number of specimens giving positive culture results each of the first (A) 60 wash solutions, and corresponding ( n ) 120 processing and (*) irradiation samples from cadaveric donors. Results from each type of specimen are batched in consecutive groups of 10.

samples were negative. Scanty colonies of the organism were isolated only after enrichment culture, suggesting that very low-level contamination of the bone was present. B. cepacia or related organisms had not previously been isolated from any sample submitted by the bone bank, nor from other grafts (heart valves and bone marrow) and DNA vaccines also monitored with similar techniques. Details of the investigation of the source of this contamination have been published previously.7 Autoclaving of deionized water in the tissue bank had been suspended shortly before the first isolate was detected, with water being taken directly from the deionizer (Elgastat UHP). Scant colonies of the same organism were isolated by filtration of 500 mL water from the deionizer. The water had been checked weekly for endotoxin activity by the LAL test (BioWhittaker, UK), but the results had been consistently negative. Commercially sterilized water is now used in all processes in the bone bank that come in to contact with bone, and the B. cepacia-like organism has not been isolated again.

Figure 2 shows the positive results obtained from each of the first 60 wash solutions (60 cultures), processing (120) and irradiation samples (120) from cadaveric donors, batched in consecutive groups of 10. There is a downward trend in growth from wash solutions, but little change in the proportion of positive cultures from processing or irradiation samples. There were no discernable changes with time in the corresponding figures for the living donors, although the numbers processed were smaller. No micro-organisms were isolated from the wash solutions, and the contribution made by the B. cepacia isolates during the period of contamination was proportionally greater.

Bacteria and fungi were isolated from various combinations of media,


and only the blood agar medium incubated at room temperature failed to recover any isolates that were not recovered from other media. Twenty- one isolates were recovered only on blood agar incubated in COz, four on anaerobic blood agar, and two on Sabouraud agar. Isolates classified as ‘significant’ were frequently recovered on only one or two media.

To date, no reports of adverse outcomes have been received from users of the bone processed during the period of contamination, although only about 50% completed record forms are returned.

Discussion

There are numerous opportunities for contamination of bone for transplant. Exclusion of live or cadaveric donors with obvious signs or markers of systemic infection will prevent use of bone that has been grossly contaminated via the haematogenous route.’ However, agonal bacteraemia (at the time of death) and spread of bacteria through the great vessels after death may be important in cadaveric donors.x Enteric bacteria, including coliforms and Clostridium spp., are common in agonal bacteraemias, probably due to loss of the barrier function of gut capillaries. Martinez et al.” found 38% of blood cultures from clinically non-septic cadavers to be positive up to 30 h after death, whereas only 8.6% of ‘beating heart’ donors were bacteraemic; identical organisms were isolated in 73% cases when both blood and marrow cultures were positive. Koneman and Davis”’ isolated bacteria from the heart blood of 53% cadavers, most commonly Escherichia roli (30%) and Klebsiella spp (11%). Bone cultures were not performed, but the patients had been dead for only up to 6 h when samples were taken. This is much earlier than harvresting of bone is carried out in the LJK, which will allow more time for organisms to multiply and disseminate. Spread of bacteria via the bloodstream would seed harvested grafts internally as well as on their outer surfaces. Negative results may be likely if only surface swabs were taken (as recommended in the guidelines of the American Association of Tissue Banks’) because surface sampling of tissue is much less sensitive than culture of biopsies in broth.” However, when internally- contaminated bone was cut and fashioned, bacteria would be released and might be detected in enrichment cultures performed after processing.

During procurement, bone may become contaminated from many sources even under operating theatre conditions.12 Mortuary air and surroundings are not clinically clean, but preparation of the operator and instruments can be as thorough as for surgical operations, and the donor’s skin can be more vigorously disinfected.

For bone wash solution cultures, we chose arbitrary cut-off levels of 50 and 100 cfu/mI, for specific pathogen and general contamination respectively. Similar levels have been used for many years by a number of North American tissue banks. No studies validating this choice appear to have been published, although we are not aware of any data suggesting they are inappropriate for bone that is terminally irradiated. Others have required


such cultures to be sterile before bone grafts are accepted for transplant,13 but when the East Anglia Tissue Bank was first established we thought this was an unrealistically stringent requirement. On the basis of our first two years experience, it is now reasonable to suggest that theatre-procured bone should be sterile, but that a background level of contamination must be acceptable for mortuary-derived grafts. It is possible that endotoxin and other bacterial products present in high concentration in procured bone may retain activity in finished grafts, and a variety of bacterial products may release cytokines from the host-graft interface, induce bone absorption and hence interfere with engraftment.14 We are at present studying the persistence of lipopolysaccharide bioactivity in experimentally contaminated grafts in an effort to set more scientifically based standards.

Application of our sampling and culture methods and use of the arbitrary standards for acceptibility described above resulted in the rejection of only one batch of bone in a two-year period, although a common source of contamination with B. cepacia was detected and eliminated. By contrast, use of standards commonly applied in North American tissue banksI would have resulted in rejection of bone from 78 of the 100 processing sessions. Until sampling techniques have been microbiologically validated, and the likely clinical significance of different levels of contamination established, it is not possible to comment upon which of the varied methods and standards in use gives the best and most cost-effective outcome. We have applied the same methods and standards to cultures taken at procurement, during processing, and before release from all tissues handled by the EA Tissue Bank, and this consistent and standardized approach greatly eases technical work and interpretation within the microbiology laboratory.

Contamination during processing in the bone bank is most likely to occur from liquids used during processing.’ Contamination may also occur during surgical implantation in the same ways that may occur during procurement in theatre. Finally, monitoring cultures may be contaminated within the laboratory to give false-positive results. Broth enrichment cultures are necessary to detect small numbers of organisms on bone grafts,” but they are prone to contamination. The assessment of significance is easier for routine clinical specimens; for example, Morris et ~1.‘~ studied 356 isolates from broth cultures of a variety of clinical samples that were not recovered on solid media, and judged 73% to be culture contaminants. It is possible that a similar proportion of the isolates in the current series were introduced during culture, although preliminary mock experiments indicated that less than 2% negative-control cultures became contaminated (data not shown).

Common laboratory contaminants of enrichment media include the same skin commensals (most commonly CNS) and environmental flora (for example, Bacillus spp.) that may genuinely contaminate bone grafts,l’ hence there are few species that strongly indicate either bone or laboratory contamination. Organisms possibly indicating a source outside the microbiology laboratory include pseudomonads (such as B. cepaciu in the current


series) which are regular isolates from wet sources in the environment, and Clostridium spp. and coliform bacteria which are commonly grown from agonal and post-mortem cultures of blood and bone marrow,” but which were uncommonly isolated from bone in our experience. Pro- cessing of duplicate samples will increase sensitivity, and we had hoped they would allow cases of laboratory contamination to be reliably recognized. However, the isolation of B. cepacia from only one duplicate sample in three of the six bone grafts implies that genuine contamination of grafts may also produce only intermittently positive cultures.

In cadaveric grafts, we noted a downward trend in the proportion of wash solutions from which any micro-organisms were isolated (Figure 2). This may have been due to increasing experience gained by procurement staff which had lead to improved aseptic technique. This trend has continued in more recent procurements (data not shown). We isolated no organisms from bone procured in theatre, and this was undoubtedly due to our culture methods deliberately not being of the highest possible sensitivity. Preliminary experiments employing more sensitive techniques suggested that organisms could be isolated from most wash solutions, but concentration with filtration or centrifugation gave inconsistent results because the wash solutions contained variable amounts of lipid and tissue.

Now we have gained experience with the techniques described in this report, we are considering altering the protocol such that irradiation samples are stored at - 20°C and only sent for culture if the corresponding processing culture gives positive results. This would avoid the difficulties encountered with the cadaveric graft with negative wash and processing cultures, but positive duplicate irradiation cultures (Table I). We suspect this represented contamination of both duplicate samples in the microbiology laboratory. Although no bacteria were isolated only from plates incubated at room temperature, psychrophilic bacterial contamination is a possibility during processing when liquids are brought into contact with the graft. Anaerobic culture uncommonly isolated bacteria that were not also grown on other media, and no obligate anaerobes were isolated. However, we found that interpretation of mixed cultures was aided by being able to inspect plates incubated under both aerobic and anaerobic conditions. We therefore intend to retain culture under anaerobic conditions and at room temperature.

Because we did not speciate or type the CNS isolates in this study, it is not possible to comment on whether identical organisms were persistently isolated from wash solution, processing and irradiation samples (Table II), which might indicate carryover of graft contamination through the stages of processing. CNS were isolated from the wash solutions of three cadaveric donations that also grew CNS from processing samples (from only one duplicate sample of each). Bacteria were isolated from 21 (33.3%) of 63 wash solutions from cadaveric donors, although in only one case was one of our criteria for rejection exceeded. In contast, no organisms were isolated from any wash solution from a living donor (Table II). This difference


may reflect post-mortem spread of organisms through the cadaver, or external contamination of the graft from the mortuary environment. Living donor bone was frozen almost immediately after resection at -2O”C, whereas cadaveric bone was stored and transported at 4°C; it is possible that bacterial multiplication occurred during transport at the latter temperature, but it is unlikely that this occurred to such an extent as to explain the observed difference. Interestingly, there was a smaller difference between the two types of donor with regard to positive processing cultures, and irradiation cultures were uncommonly positive from either source (see Table II). Positive processing cultures may result from carryover of organisms from the harvested graft, and internal contamination of the bone may be relatively more significant at this stage. Alternatively, contamination may arise within a bone bank, as was found with the B. cepacia isolates. We are considering changing our procedures so that bone samples would be added to universal containers of broth within the clean room suite before they were sent to the microbiology laboratory, and it is possible that use of commercially-sterilized culture media might further reduce the culture- positivity rate.

Contamination of water deionizers is a well-recognized problem in hospital infection control practice,” and in a report analogous to our own experience, Mermel et aZ.14 described contamination of four grafts with Comomonas acidovorans and Pseudomonas sp. from a water-bath. We believe that continuously assured sterility is essential for water that comes in to contact with materials for patient implants. Although it would be convenient to rely on audited standard operating procedures to assure the microbial safety of transplanted bone within tissue banks, our experience leads us to recommend that culture monitoring is required in addition, and other publications attest to the value of culture accreditation of tissue grafts being done by external agencies.” Although the only report we can find of bacterial infection of an implanted bone graft clearly acquired from the donor or from contamination within a tissue bank occurred many years before donor screening was developed,” clinical infection via a variety of other tissue transplants has been reported.‘*‘*~** In addition, longitudinal surveillance of culture results is necessary to detect recurrent isolates that might indicate a common source for low-level contamination that was not being consistently detected.’ Jumaa and Chattopadhyay23 provide useful checklists for recognizing and investigating sources of contamination of blood cultures which are largely applicable to tissue bank cultures. After investigating the cause of the problem, we chose to release the batches of bone that had been contaminated during processing with B. cepacia because the contamination was at a very low level, endotoxin was undetectable in the deionized water, and all post-irradiation cultures were sterile.

We recommend that appropriate microbiological testing of bone for transplant should be performed at the time of its entry to tissue banks and after processing. This will not only protect recipients from infection, but


also pro\Tide a valuable process control, and it is likely to be particularl) important in newly established tissue banks.

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References

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Microbiological monitoring of bone grafts: two years' experience at a tissue bank

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