Poster 1:
New therapeutic approach in the treatment of severe radiation burn: surgery and local stem cell therapy.

• Prat M.,
• Bey E.,
• Brachet M.,
• Trompier F.,
• Ernou I.,
• Boutin L.,
• Gourven M.,
• Tissedre F.,
• Créa S.,
• Ait Mansour C.,
• de Revel T.,
• Carsin H.,
• Gourmelon P.,
• and Lataillade J-J.

The therapeutic management of severe radiation burns remains today a challenging issue. The conventional surgical treatment (excision and skin autograft or flap) often fails to prevent unpredictable and un-controlled extension of the radiation necrotic process.

We report here our second experience of therapeutic management of a radiation accident victim combining surgery and cellular therapy using autologous Mesenchymal Stem Cells (MSC).

The patient presented a very severe arm radiation burn which was treated by several surgical times: iterative excisions, skin graft, latissimus muscle dorsi flap and forearm radial flap). Local autologous MSC were administrated as an adjuvant to improve the surgical approach. The clinical evolution (radiation pain and healing progression) was favourable and no recurrence of radiation inflammatory waves was observed during the eight month patient's follow-up suggesting that MSC act as "cell drug" in modulating radiation inflammatory processes.

These results open new prospects in the medical management of severe radiation burns.

Poster 2:
Cell Regeneration Therapy as an adjunct to healing in recalcitrant wounds.

Cairns S.A., Leaper D.J., Harding K.G.

Department of Wound Healing, Cardiff University, Wales.

Background:

Chronic wounds have for many centuries represented a major source of morbidity and mortality in patients. In the modern era we realise that these wounds also produce a major psychological impact to the patients, and a financial burden to the healthcare systems around the world.

Ever since the first skin grafts were attempted in India around 2000bc for the reconstruction of punitatively amputated noses, the surgical profession and medical sciences have been searching for the panacea to treat chronic wounds. Yet as our understanding of the aetiology of chronic wounds deepens we realise that the varied nature of the wound pathogenesis means there will never be a "one cure for all" treatment. In this way we can see that a split skin graft is certainly not the optimal option for many patients, and that alternatives must be found.

Method:

In two selected patients with chronic static laparotomy wounds we demonstrate the successful use of a biological tissue regeneration therapy (Apligraf®) in the management of these recalcitrant wounds.

Conclusion:

In carefully selected patients under the care of a Multidisciplinary Wound Healing Team, the use of cell based tissue regeneration products may provide a valuable adjunct to standard wound healing regimens.

Poster 3:
Deep venous incompetence in patients with diabetes mellitus: a case control study.

Starla Yarde, Puja Patel, Geoff Roberts, and Raj Mani

Vascular Laboratory Services, Southampton University Hospitals Trust, Tremona Road, Southampton, UK.

Introduction:

Venous hypertension has been suggested to be a risk factor for diabetic foot disease. This is considered to the effect of autonomic neuropathy though this appears to be based on clinical evidence. It has also been reported that diabetics with neuropathy have impaired venous function and haemodynamic dysfunction. Deep venous incompetence causes venous hypertension and haemodynamic changes. The prevalence of deep venous incompetence in the diabetic population is largely unknown. This aim of this study was to determine the prevalence of deep venous incompetence in the diabetic population in Southampton since as diabetic foot disease is a major problem.

Methods:

This study was a case-controlled study at the Southampton General Hospital. The study included two groups of subjects recruited from Diabetic Foot Centre that is a referral point for diabetic care including foot disease. Patients were subdivided in to groups.

• Group 1 - The diabetic patient with frank ulcers or healed ulcers.
• Group 2 - The diabetic control subject with no foot ulcers.

Each patient underwent a duplex ultrasound scan to measure venous reflux for deep venous incompetence and laser Doppler flowmetry (LDF) scans. LDF scans were done to determine the arterio-venous response (AVR) and the venous refilling time (VRT) respectively. The data were analysed using the Pearson Chi square, Fisher's exact and the Mann Whitney U tests. The study was conducted with Ethics approval and prior informed consent of patients.

Results:

75 subjects were recruited and tested.

64% and 70.7% of diabetic subjects had deep venous incompetence in their right and left legs respectively which is statistically significantly greater (p<0.05) than a previous report.

42.7% and 49.3% of subjects had a reduced VRT in the right and left legs respectively.

30.7% and 33.3% of subjects had loss of the arterio-venous response in the right and left legs.

The presence of symptoms was also recorded during the study.

Discussion:

These data permit the conclusion that here is a high prevalence of deep venous incompetence in subjects with diabetes mellitus in our practice. Given that venous incompetence is reported to be a risk factor for diabetic foot disease and the argument that venous disease impairs wound healing, should we be treating this aspect of the circulation in the diabetic population who are at known risk of foot disease?

Poster 4:
Intensive treatment of wounds in lower limb amputation surgery can save a leg!

C. Naves, V. Brehm, P. Steenvoorde, P. P. Hedeman Joosten, S.A. da Costa.

Department of Surgery, Rijnland Hospital Leiderdorp, The Netherlands.

Trans-tibial amputations have less co-morbidity than trans-femoral amputations. There is more stability when sitting, rehabilitation is easier and, most important of all, patients will preserve more of their self esteem.

However, trans-tibial amputations wounds have a higher risk of infection than trans-femoral amputations. Infection of the trans-tibial wound results in longer hospital stay, another operation and can even lead to a trans-femoral amputation.

In our clinic we have a very intensive treatment scheme for amputation wounds. This includes, amongst other techniques, frequent inspection of the wounds, Maggot debridement therapy and Vacuum Assisted Closure therapy (VAC). Using this scheme, our hypothesis is that we prevent patients from undergoing a trans-femoral amputation.

In a prospective study, we investigated all lower limb amputations from January 2003 until January 2007. During this period, 67 patients underwent a trans-tibial amputation. 51 of these 67 patients (76%) went home with a healed wound, 6 patients died within 30 days of surgery and in 10 patients conversion to a trans-femoral amputation was necessary.

When comparing these outcomes to the literature on lower limb amputations, we find that our intensive treatment scheme pays off. In our clinic we relatively perform more trans- tibial amputations than primary trans- femoral amputations. Furthermore, our trans- tibial amputation wounds had a significantly higher healing rate than the wounds described in the literature (76% versus 44-62%). Also, a smaller percentage of trans-tibial amputations is converted to a trans-femoral amputation. These results demonstrate that it is better to use an intensive treatment scheme in order to save a leg, since it has a significantly higher success rate!

Poster 5:
Platelet Rich Fibrin (PRF) for hard to heal ulcers in patients with diabetic feet.

Caroline Naves,¹ Pascal Steenvoorde, ^1,2 Louk P, van Doorn MA²

1. Department of Surgery, Rijnland Hospital Leiderdorp
2. Rijnland Wound Clinic, Rijnland Hospital Leiderdorp,The Netherlands.

Platelets play two important roles in wound healing: hemostasis and initiation of wound healing. After platelet activation and clot formation, growth factors are released. In ordinary blood, the number of platelets is 0.2x 106/ul. In contrast, it is >1.0 x 106/ul in platelet-rich plasma.

Autologous growth factors from concentrated platelet suspensions have been used to treat wounds for years. The use of platelet-derived wound healing formulae delivered in a crystalline collagen carrier was first published in 1986.

The Vivostat® System is a medical device for the preparation of an autologous fibrin sealant from 120ml of the patient's blood. The system is fully automated and microprocessor controlled and is made up of three components: an automated processor unit, an automated applicator unit, and a disposable, single-patient-use unit, which includes a preparation set and a Spraypen applicator.

This pilot study of very hard-to-heal ulcers investigated whether treatment with autologous platelet-rich fibrin is feasible and to see if wound healing is improved. We would like to discuss our results of application of PRF (Vivostat PRF, Birkeroed, Denmark) in 8 treated patients with a diabetic foot ulcer.

Between September 2006 and November 2007 we recruited 18 patients with chronic, hard to heal ulcera. All wounds were located on the lower limb. In our population of 18 patients, there were 8 patients with a diabetic foot ulcer. 9 wounds were included in this study. (One of the patients had 2 wounds on the lower limb which were treated simultaneously). The population consisted of 6 males and 2 females with a mean age of 55.3 years. (range 38-70). The mean wound duration before treatment was 7.5 months (range 1-24).

The patients received a total of 23 treatments. In total, with a short follow up, (1-24 months), over 60% of the wounds have closed. 2 wounds were smaller than 1/3 of the initial size, although these are estimates only.

Poster 6:
A comparative study of the influence of different pressure levels combined with various wound dressings on negative pressure wound therapy (NPWT) driven wound healing.

Rey Paglinawan

Medela AG, Medical Technology, Lattichstrasse 4b, Baar, Switzerland.

Introduction & Aim:

To present date, although a number of studies have been conducted to support the important role of NPWT in wound healing, none have investigated the optimal pressure level intensity and manner of delivery of negative pressure together with various combinations of existing dressing technologies. This study compared different commercially available dressing types in combination with a NPWT system used at various pressure levels. Wound healing progression from these various parameters was subsequently assessed.

Methodology:

Five pigs (Landras type) were allowed to acclimatize to the housing conditions prior to all procedures. The animals were sedated, shaved and scrubbed for surgery and transported to the operating room. Full thickness wounds extending to the fascia over the deep back muscles (3.5cm x 3.5cm, maximal 10 per animal) were excised on each pig. Different commercially available materials (foam, gauze, silver, textiles) were trimmed to the exact size of each wound. Each wound site was sealed with a thin air-permeable adhesive film. The dressing system is then connected to a vacuum source which was set to a different level of negative pressure (10, 20, 30 kPa).

Result & Conclusion:

Morphological and histological observations of our wound porcine model indicate that healing is taking place independently from the type of dressing used. Thus, providing insights that there is currently not one type of dressing which is clearly outperforming the others when used in combination with negative pressure therapy on porcine wound models.

Poster 7:
Correlation of physical properties and dressing design.

J.M. Dillon, J.V. Clarke, A. Nicol

Department of Orthopaedics, Dumfries & Galloway Royal Infirmary, Bankend Road, Dumfries, UK.

Aims:

Wound problems such as blistering are common following total knee replacement (TKR) surgery. The physical properties of the wound dressings may be responsible for this. They may not stretch to accommodate skin movement and consequently transmit high levels of shear force to the skin.

Skin movement of TKR wounds was quantified and compared with the material properties of a hydrocolloid, adhesive and two occlusive film dressings.

Methods:

TKR wound measurements were taken at 0°, 30°, 60° and 90° knee flexion in 85 patients. Percentage change in wound length at each flexion level was calculated.

The material properties of the dressings were quantified using an Instron 5800 uni-axial testing machine. Strain was calculated using grip to grip measurements. Three samples of each dressing were tested.

Results:

The traditional dressing showed very high loads for less than 5% strain. The other three dressings extended to at least 25% strain with less than 10N load.

Figure 1: Dressing material properties showing load with strain.

The hydrocolloid and both occlusive dressings extended the required amount to accommodate skin movement (28% strain) with loads of 7.7N, 8.9N and 6.5N respectively. The adhesive dressing extended to <10% of the required amount before reaching the limit of the 100N load cell.

Discussion:

TKR wounds demonstrate dynamic morphology and strains of over 20% with normal knee flexion. Hydrocolloid and some occlusive dressings exhibit suitable material properties to accommodate this skin movement but traditional adhesive dressing do not. This may explain the high blister rates with some dressings.

Poster 8:
LASER stimulation on fibroblasts WI-26 based on photodynamic processes.

Fernando L. Primo¹, Andreza R. Simionia,², Bernard Coulomb² and Antonio C. Tedesco^1,2

1. Departamento de Química, FFCLRP, Universidade de São Paulo, Ribeirão Preto, 14040-901 (Brasil).
2. Inserm U849. Université René Descartes - Paris 5, Paris (France).

Phthalocyanines are interesting compounds for use in Photodynamic Process (PDP) considering their high absorbance (680 nm), with optimal tissue penetration by light. Chloroaluminium-phthalocyanine (AlClPC) is a photosensitizer drug applied in the treatment of cancer and others no-oncological diseases by PDP assay. The main goal of this work was to develop and study an AlClPC-nanoemulsion to be used in combination with low energy LASER to modulate cellular growth of fibroblasts.

WI-26 fibroblasts were used in vitro biostimulation studies in monolayer after light application. The cells were incubated with AlClPC-nanoemulsion for 30 min in the dark, with later irradiated at 670nm; at different energy doses (24h and 48h) after nanocarrier treatment. Cellular viability was measured 3 and 5 days after irradiation using a MTT classical assay. Scratching experiments to mimic the wound repair have been done using the same protocol.

Irradiation by low energy (1.0 to 5.0 J) stimulated cellular growth in a dose-dependent manner, while the energy of 10J showed a negative response for this biostimulation. The results also showed that the biomodulation was time-dependent, higher with a delay of 48h between dye and light application.

This protocol of PDP will know be used on in vitro reconstructed tissues to better assess its effect on the wound healing process.

Poster 9:
BITECIC (Biomaterials and Tissue Engineering Centre of Industrial Collaboration) - A collaborative approach for creative partnerships.

Claire Green

BITECIC Ltd, Thorpe Park, Leeds United Kingdom.

Aim:

To provide an insight into the BITECIC model and its methodology for fostering productive collaboration. BITECIC (Biomaterials and Tissue Engineering Centre of Industrial Collaboration) was established in recognition of the need for a collaborative approach to enhance the development of advanced clinical treatments and medical devices in wound care and other medical specialisms. Developing creative partnerships by bringing together diverse expertise from an academic, clinical and commercial perspective is at the heart of BITECIC's way of working and contributes to the development of novel therapies that meet clinical and commercial expectations.

This collaborative approach will be of particular value in wound care, which faces diverse and complex clinical and commercial challenges. The effectiveness of working in partnership with industry, clinicians and academics was illustrated by a recent project to determine the needs and opportunities in the area of chronic wound infection. This example will be described in order to provide an insight into the BITECIC model and its methodology for fostering productive collaboration.

BITECIC is committed to the development of solutions to real clinical needs. In order to support this, BITECIC is working in collaboration with the Healthcare Technologies Knowledge Transfer Network (Healthtech KTN) to develop the Statements of Clinical Need (SOCN) initiative. This provides the infrastructure to connect the industrialists, healthcare professionals and researchers who comprise the UK Health Technologies community and to facilitate communication between these specialist groups. The SOCN project will be described and delegates encouraged to participate.

Poster 10:
The effects of nicorandil on wound healing (case series).

A.D.Riddell, D.J. Leaper. K.G. Harding

Wound Healing Research Unit Cardiff University, United Kingdom.

Introduction:

We would like to report a series of nicorandil induced non-healing wounds seen over the past year.Nicorandil is a nicotinamide ester, and derivative of synthetic nicotine. It functions as a potassium channel activator. Uniquely Nicorandil has a nitrate group in its structure, adding venodiliatation to its effects. Thus causing increased blood flow in the coronary arteries and decreasing preload, the effects combining to form a highly effective anti anginal. The association of nicorandil use and oral/anal ulceration is now fairly well recognised. Less well known are nicorandil's association with para-stomal ulceration, and indeed its association with ulceration of the GI tract from mouth to anus. More recently there have been increasing reports of nicorandil-induced ulceration in parts of the body distant to the GI tract. We report a series of cases seen in our wound healing clinics, consisting of, patients, with lesions ranging from post surgical wounds to leg ulceration.

All these ulcers had a common clinical appearance: there was no visual evidence of healing, all lesions were extremely painful, and all healed rapidly following withdrawal of nicorandil.

Objectives:

The aim of this work was bring to a wider audience our experiences of nicorandil's association with non healing wounds.

Methods:

Patients included in the study were those who have presented to the Wound Healing Outpatient Clinics' of the Wound healing research unit at Cardiff university over the last 18 months. All patients were taking Nicorandil, and had typical non healing painful wounds, which have resolved or moved rapidly towards resolution following exclusion of Nicorandil from their treatment.

Results:

The mechanism and incidence of the non-healing associated with Nicorandil is currently unknown, and may or may not be associated with the properties of arterial and venous dilatation. It had previously been assumed that the nicorandil itself caused the ulceration being seen in the GI tract. We would hypothesise that the effects of nicorandil (either directly or via metabolites) is not in causing Ulceration/Wounds, but in preventing the normal healing mechanisms of healing in a pre-existing wound. Thus explaining why initially non-healing GI tract ulcers were observed (as this is a common site of injury/ulceration), but since then non-healing wounds, in other sites, and even post surgically have been observed.

Conclusion:

As the use of nicorandil increases these painful, debilitating, costly, and yet easily treatable wounds will become more common and a high index of suspicion is needed in any patient taking nicorandil, with any non healing wound.

Poster 11:
A prospective trial of near-infrared spectroscopy (NIRS) as a contionuus non-invasive method of flap monitoring following breast reconstruction.

Cairns SA^1,2, Whitaker IS¹, Barrett MD¹, L Hiew¹, Boyce DE¹, Cooper MACS¹, Leaper DJ²

1. Department of Plastic, Reconstructive and Burns Surgery, Welsh Centre for Burns and Plastic Surgery, Swansea, UK
2. Department of Wound Healing, Cardiff University, Cardiff, United Kingdom

Background:

In the UK, following mastectomy for Breast cancer, patients are offered a choice of reconstructions, up to and including myocutaneous flap reconstruction. Arterial or venous embarrassment represents a major cause of complications, often requiring intervention or resulting in total flap failure. A system to recognise impending problems would improve salvage rates. Near Infra-red spectroscopy (NIRS) has previously been shown to be a reliable indicator of tissue status in trauma.

Objective:

This pilot study examines the use of NIRS as a tool to assist the clinician as a means of monitoring myocutaneous flaps following oncological reconstructive surgery.

Methods:

Nineteen patients having myocutaneous flap reconstruction, either as an immediate or delayed reconstruction, were enrolled into the study. Measurements were made using the Inspectratm St02 monitor (Hutchinson®), measurements of tissue oxygen saturation (St02) and Total Haemoglobin Index (THI). Measurements were taken prior to, during, and after the surgery; with post-operative monitoring occurring continuously for 72 hours. Data were correlated with clinical outcome measures.

Results:

Out of the nineteen patients, there were two complete flap failures and four complications requiring intervention. In these six cases, problems were identified by either a significant rise in the THI or drop in the St02. One patient's data were unrecordable in recovery, and the patient was therefore returned to theatre immediately for a salvage procedure, which was ultimately successful.

Conclusion:

The Inspectra™ St02 monitor (Hutchinson®) has proved to be a useful aid in the clinical setting. While not replacing the need for experienced and rigorous nursing care, we believe it can be a valuable and objective means of assessing flap viability.

Poster 12:
Wound diagnostics: can a single molecular marker concur with an expert's multifactorial assessment of wound healing status?

Sandra Hemmington¹, Paul Davis¹, James Schouten¹, Mark Burnapp¹, Douglas Queen^1,2, Pat Coutts²

1. Mologic Ltd. UK
2. Toronto Wound Healing Centres, Canada

Aims:

Diagnostic tests are needed to improve outcomes and health economics in wound care. Ideally, a healing status indicator should provide the highest level of diagnostic accuracy (normally available only from specialised centres of excellence), regardless of the carer's expertise.Mologic has developed a novel rapid protease assay with an indicator molecule biased towards MMPs 9 and 8, to indicate wound healing status. The aim of this study was to assess correlation between the test result and expert clinician judgement.

Methods:

Fluids from 10 randomly selected wounds judged to be healing were collected onto swabs, which were stored and shipped to the UK at -80°C. Fluids from another 9 randomly selected wounds judged to be non-healing were processed identically. Samples were coded to ensure blinding to wound identities. Extracts of the samples were assayed by conventional procedures for total protein, MMP 8 and MMP 9 (commercial immunoassays) and with zymography, as well as by the non-optimised prototype novel rapid assay. The results of all the tests were compared with each other and with the expert clinical judgement.

Results:

The prototype rapid assay results did not correlate with conventional tests, but they did correlate with the expert judgement. 7 of 9 "non-healing" wound samples were protease positive. 7 of 10 "healing" wound samples were protease negative and another 3 had reduced protease activity. Under these particular prototype conditions/thresholds and clinical assessment criteria, a sensitivity of 77% and specificity of 80% were achieved.

Conclusion:

Appropriate assays can be tuned to correlate well with expert clinical judgement. Further developments will improve correlation.

Poster 13:
Development of a novel growth factor formulation to maintain serum-free and feeder cell-free culture of human embryonic stem cells.

Sean Richards, Kerry Manton, Zee Upton

Tissue Repair and Regeneration Program, Institute of Health and Biomedical Innovation, Queensland University of Technology, Kelvin Grove, Brisbane, Australia.

To realise the full clinical potential of human embryonic stem (hES) cells, culture methods that do not involve animal products or purified/non-defined factors must be utilised. This requires removal of animal feeder-cells, serum and any other animal-derived products.

We have utilised a subtractive proteomic approach to analyse the components in culture medium that are produced by the feeder cell layer in the presence and absence of hES cells. We have subsequently used this information to add select candidate factors in recombinant form to develop a serum-free and feeder cell-free culture system for hES cells. We have successfully cultured hES cell lines (BGV01 and HUES2) for over 30 passages using this new serum-free and feeder cell-free formulation. The medium contains low osmolarity DMEM, glutamax, LiCl, ß-mercaptoethanol, Lipid concentrate, Trace elements, Activin-A, bFGF, Albucult™ (recombinant human albumin, Novozymes Biopharma UK Ltd.) and an in-house recombinant chimeric protein called VitroGro® (composed of domains of vitronectin linked to IGF-I).

We have shown by immunofluorescence and FACS that hES cells cultured in this serum-free and feeder cell-free culture system express the undifferentiated cell-surface markers SSEA4, TRA1-60 and TRA1-81 and the intracellular marker Oct4. Real-time PCR analysis has revealed that the expression of UTF1, SOX2, FOXD4, OCT-4 and Dppa by these cells does not change more than two-fold compared to cells cultured in the presence of serum and feeder cells. Interestingly, hTERT is down-regulated four-fold and REX1 is down-regulated 75-fold in the cells grown in the serum-free and feeder cell-free system. REX1 has recently been linked to increased differentiation potential; hence the down-regulation of REX1 in these cells may demonstrate their stable pluripotent status. We have recently injected the cells into SCID mice to observe their ability to form teratomas in-vivo and will also present data from these studies.

In conclusion, our serum-free and feeder cell-free media formulation supports the undifferentiated growth of hES cells for over 30 passages and allows hES cells to be cultured in xeno-free, fully defined, synthetic media. The media developed in our laboratory contains very few growth factors and these are used at concentrations much lower than those previously published by others. This in turn substantially reduces the cost of culturing these cells and makes large-scale hES culture for therapeutic applications a realistic possibility for the first time.

Poster 14:
Exploring the application of ultrasound technology in pressure ulcer prevention - An international multidisciplinary team study.

A. Moorhead¹, C. Adams¹, M. Stinson¹, A. Porter-Armstrong¹, E. Gardner¹, J. Donnelly², S. Deegan³, J. Nixon⁴, D. Bader⁵, C. Lyder⁶

1. Health and Rehabilitation Sciences Research Institute, School of Health Sciences, University of Ulster, Northern Ireland;
2. Belfast Health and Social Care Trust, Royal Hospitals, Belfast, Northern Ireland;
3. Northern Ireland Medical Physics Agency, Northern Ireland;
4. The Clinical Trials Research Unit, University of Leeds, UK;
5. Department of Engineering, Queen Mary, University of London, UK;
6. School of Nursing, University of Virginia, USA.

Introduction:

High frequency ultrasound (HFUS) can create real-time two-dimensional images of internal structures to examine the first few centimetres depth of sub-dermal soft tissue, thus may have the potential to detect tissue changes in pressure ulcer prevention. However, there is no evidence on the repeatability of HFUS. This study aimed to investigate the inter- and intra-rater repeatability of HFUS scanning.

Methods:

In a laboratory based study, 24 healthy subjects (21 females, 3 males; 32.08 ± 12.25 years; BMI 24.94 ± 5.57) participated on two occasions, one day apart. Consenting subjects had points marked (day 1 only) on both heels (lateral, posterior and medial aspects) and seating interface (coccyx, and left and right ischial tuberosities) and scanned by a researcher, then repeated by a second researcher using a HFUS scanner. Both researchers were blinded to each other's scanning. HFUS images were quantitatively and qualitatively analysed blindly by two assessors.

Results:

Qualitative visual analysis showed almost perfect agreement between two assessors (0.88 kappa co-efficient). Intraclass correlation coefficients (ICCs) conducted on pixel intensity summation quantitative results showed low inter- and intra-rater repeatability (25% moderate or high ICCs; ICC ≥0.6).

Conclusions:

Although quantitative analysis showed low inter- and intra-rater repeatability, qualitative analysis showed better agreement. In practice, clinicians qualitatively read the images, and visual analysis appears to be the gold standard in HFUS interpretation. Quantitative methods of analysing HFUS images require further exploration by industry, scientists and clinicians.