Intra-operative tissue sampling and microbiological analyses during minor lower limb amputations in patients with diabetes are poorly reported and difficult to interpret


1 Russells Hall Hospital, Dudley Group NHS Foundation Trust, Pensnett Road, Dudley, UK
2 CT1 Vascular Surgery, Russells Hall Hospital, Dudley Group NHS Foundation Trust, UK
3 SpR Vascular Surgery, Russells Hall Hospital, Dudley Group NHS Foundation Trust, UK
4 Consultant Vascular Surgeon, Russells Hall Hospital, Dudley Group NHS Foundation Trust, UK

Address for correspondence: Mohamed A Gulamhussein
SpR Vascular Surgery, Department of Vascular Surgery, Russells Hall Hospital, Dudley Group NHS Foundation Trust. Pensnett Rd, Dudley DY1 2HQ, UK


Diabetic foot disease (DFD) is a leading cause of acute sepsis and has long-term consequences for patients. It poses a strain on health resources in both the developed and developing world, with a significant impact on patient quality of life due to the associated complications of DFD and the often multiple interventions required to control infection and preserve limb tissue. Although there is evidence in the literature regarding early detection and prompt management of this debilitating condition, there is little structured evidence on how to gain accurate tissue sampling with processing to allow targeted antimicrobial therapy from minor amputations where bone cultures have been sent.

Methods: A literature review was conducted to establish the publications on intra-operative bone sampling and processing taken during diabetic foot minor amputations and the pathways described for processing sample acquisition.

Findings: Thirty papers were identified which highlighted some of the processes involved in the procurement of intraoperative tissue samples. No published paper reported a complete pathway for the ascertainment of samples, transfer and processing of these specimens.

Conclusion: There is no published consistent pathway published for procurement of intra-operative diabetic foot specimens, for their storage, transportation and processing. Without documented, reproducible processes, it is difficult to interpret published results. This makes planning for targeted antibiotic therapy more difficult.

Br J Diabetes 2022;22:78-81

Key words: processing, intra-operative, diabetic foot, tissue


903 Gulamhussein Figure 1

It is estimated that 10% of patients with diabetes will have a foot ulcer during their lifetime.1 This can lead to serious consequences such as major lower limb amputation. People with diabetes, when compared to the general population, are twice as likely to have amputations later in their lives.1 Mortality following diabetic foot amputation is 70% within five years and 50% following diabetic foot ulceration.1 In the UK in 2015, it was estimated that more than 135 minor and major diabetic foot amputations were carried out per week, more than 6,677 per annum.2 DFD is associated with substantial cost implications. Annual spending on diabetic foot care and amputations is estimated at between £837 million and £962 million.3

Digital amputation is a commonly performed procedure for foot salvage in the case of diabetic forefoot sepsis to drain infection. Recommendations exist on the type of specimen (bone or tissue) to take but guidance on the procurement of those specimens, transportation and processing is less well documented.1,4 Difficulties in understanding the sampling and processing pathway could have a negative impact on treatment optimisation in the pre-, peri- and post-operative period. This may result in delayed wound healing, poor antibiotic stewardship, increased rates of re-admissions or further surgery, leading to possible sub-optimal care to this cohort of patients.5

The aim of this literature review is to document the reporting of the techniques deployed in bone sampling of patients who require diabetic foot minor amputations. The review looks at the process of sample acquisition, sample storage, transportation and processing. This not only highlights the literature that has been published but also portrays the ease of data interpretation to optimise patient care. Fundamental steps in sample attainment and processing, along with confounding factors, are highlighted in Figure 1.


A review of electronic databases (PubMed, MEDLINE and EMBASE) was conducted with the aim of identifying papers which included bone sampling in patients who had minor/digital or forefoot amputations due to complications of diabetes. The search included papers up until 20th February 2021. Relevant search terms were used to identify associated papers. The search criteria used were: “(bone biopsy OR bone sample OR bone culture) AND diabetic AND amputation AND technique NOT percutaneous”

The results of these searches are summarised in Figure 2. The titles and abstracts were screened for relevance to our purposes and two of the authors (RS and HT) further examined the publications and assessed the relevant articles based on the inclusion and exclusion criteria.

All studies published which signposted how bone sampling was performed in patients who underwent minor amputations (fore- foot, hallux, single/multiple digits) were included. Publications were excluded if sampling was performed percutaneously, if there was no mention of surgical intervention or documented surgical technique and if abstracts were not available in English. All papers were then reviewed by two of the remaining authors (MG and MW) independently to ensure adequacy of the included studies.

903 Gulamhussein Table 1

903 Gulamhussein Table 2903 Gulamhussein Figure 2


Results from our search criteria yielded a total of 37 papers (Appendix 1 below). However, 30 studies were included in our analysis as there were six review articles and one systematic review requiring extraction. These are highlighted accordingly. The majority of these papers (21, 70%) were cohort series. All studies emphasised the importance of accurate bone sampling techniques for robust antimicrobial treatment in diabetic foot sepsis.

Three (10%) studies describe preparation techniques prior to sampling taking place, whilst seven (23.3%) papers described their intra-procedural sterility techniques (e.g. change of gloves, change of instruments). Twenty-five (83.3%) studies declared that a form of tissue specimen (bone, soft tissue or both) was taken intraoperatively but only 27 (90%) of the studies reported the type of analysis performed to identify the causative organism and rate of residual disease. Six (20%) studies did not specify whether any tissue sample was taken intra-operatively. These studies did not clarify whether treatment was therefore based on standard antimicrobial guidelines for diabetic foot disease. Twelve (40%) studies mentioned that a form of bone was sampled but did not specify the type or whether the site was infected or clean. All results are shown in Tables 1 and 2.

Eight (26.6%) of studies included type of incubation media use and six (20%) studies identified the control temperature and storage of samples prior to processing; however, none of the studies specify the exact transportation methods, timing or medium used once the specimens had been retrieved intra-operatively.


903 Gulamhussein Key Messages

Diabetic foot disease is a growing pandemic which requires good data to treat effectively. Vascular surgeons, diabetologists, diabetic podiatrists, microbiologists, radiologists and other members of the MDT all have a crucial role to play in the management of this complex issue.1 Minor amputation techniques have been poorly taught and poorly understood for some time.6 The authors have concerns that the process of sampling is being overlooked when it comes to gaining accurate and useful information. No core outcome set could be identified for diabetic foot disease. Recently published work by the authors would suggest that DFD sampling reporting in the medical literature is heterogeneous and leads to findings that cannot be interpreted or reproduced with ease.The fact that minor amputation for foot sepsis in the presence of diabetes with or without the presence of osteomyelitis is performed by junior surgeons, often out of hours, in the UK remains of concern. This stimulated the authors to look at the published literature on the techniques reported for bone sampling as recommended by NICE (NG19) in the population undergoing minor amputations.1,6

The published literature on minor amputation with sampling for diabetic foot disease is based around small series of patients in single centres.4,5 These studies fail to report the process of sampling adequately and make interpretation of results difficult, as described previously. Authors who have undertaken systematic reviews on similar subjects report the concerns with sampling techniques and therefore accuracy of results that we have raised through this publication.5,8 Diabetic foot amputation is often performed in septic patients bearing tissues with gross tissue destruction whereas most published studies are based around chronic diabetic foot ulceration, which is clearly a different situation.5,9,13 Understanding the differences between the success and process of sampling between the two situations will help benefit planning and delivery of services in the future.

Atway et al and colleagues in 2012 and Schmidt et al in 2020 emphasized that the presence of residual osteomyelitis or positive bone margins is associated with poor outcomes, with higher rates of residual infection leading to morbidity.4,9 Their studies emphasised the importance of debridement technique and robust proximal bone sampling to guide optimal antimicrobial therapy and achieve better outcomes. This sampling can often be supported by effective imaging, such as recommended by Cohen et al, and by the use of MRI.10 Accurate sampling is vital for targeted antibiotic therapy. Authors sporadically report the use of culture mediums to support sample transport, suggesting more accurate yields from their specimens to improve the accuracy for targeted antibiotic therapy, but there are only a few small-scale studies in the literature.11 This concerns the authors, as the growing pandemic of diabetic foot disease will require a concerted effort backed by reproducible data and robust guidelines to maximise good outcomes for patients.

It is clear that the literature supports the use of intra-operative bone specimens in diabetic foot treatments to guide accurate diagnosis,5,7 but as can be seen from this study the techniques deployed to gain these samples are poorly reported, with no standard of care present in the literature or in the NICE NG19 guidelines. Moreover, standardised methods of immediate intra-operative culture storage and transportation are not specified at all, which leaves uncertainty in a crucial part of treating this complex disease. Public Health England in 2015 published a standard operating procedure to guide specimen storage and processing time. The recommendation was for transfer of specimen collection to the laboratory within two hours. We have been unable to validate from the published literature whether this guidance is being followed. In practical terms, this can be difficult to deliver in the theatre or clinic environment due to logistical difficulties such as out-of-hours working.6,12 At present, there is a lack of evidence to show that rapid transfer times to the laboratory or short turnaround times have any long- term benefits on the outcomes after minor amputation where bone sampling has been performed. Further investigation into the process of sampling technique and specimen handling should be considered to gain accurate yields from bone sampling in minor amputation. Future cohort studies are required to see if this leads to beneficial outcomes to patients. Appropriate sampling guidelines backed by appropriate education for clinicians should be developed to guide future sampling, transfer and processing techniques to maximise benefit from therapies.6,12,13


This literature review concludes that there is no standardised method for sampling, transportation or processing of bone biopsy specimens taken for those DFD patients who require minor amputation. Considering the high prevalence of this condition and requirements for surgical interventions, a robust pathway and standardised technique must be ascertained and described routinely in the medical literature to allow for the optimal utilisation of antibiotic therapy in this disease.

Conflict of interest None.

Funding None.


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Appendix 1. Intra-operative tissue sampling in minor lower limb amputations is poorly reported.

(All papers yielded in our qualitative review)

903 Gulamhussein Appendix 1a

903 Gulamhussein Appendix 1b

903 Gulamhussein Appendix 1c

903 Gulamhussein Appendix 1d

903 Gulamhussein Appendix 1e

903 Gulamhussein Appendix 1f


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