Imaging the superficial lymphatic system of the lower limb after soft tissue injury.

Author: Malou Van Zanten

Van Zanten, Malou, 2016 Imaging the superficial lymphatic system of the lower limb after soft tissue injury., Flinders University, School of Medicine

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Severe open fractures due to high energy trauma require soft tissue reconstruction with local, regional or free tissue in addition to the fixation of bone injury. Oedema, both within and surrounding the reconstructed site, can present acutely in the post-surgery setting. However, in some patients the swelling fails to resolve and chronic oedema develops. This is lymphoedema, when the lymphatic system is in a state of failure, either due to its inability to regenerate in the wounded area or its inability to handle the increased load imposed in the post-traumatic period. Lymphoedema is the accumulation of fluid in the tissues. As lymphatic failure progresses, lymphoedema worsens, resulting in visible swelling, mobility issues and there can be associated discomfort, heaviness and pain. While lymphoedema is commonly recognised as secondary to breast cancer and its treatment, it can also occur after trauma with extensive soft tissue damage or loss. A review of the literature showed no current best practice protocols are available for traumatic lower limb lymphoedema.

New lymphatic imaging techniques, such as those explored in this thesis which are focused on lymphatic function may however provide an improved understanding of lymphatic failure in these trauma related conditions. With this there is a possibility to identifying the reasons for poor regrowth and inosculation of lymphatic channels, or the effect of increased loads on the existing lymphatic system. The imaging technique which forms the core of this thesis involved the contrast agent Indocyanine Green (ICG), in combination with Near Infra-Red (NIR) imaging system. Its use allows a minimally invasive real-time image of the superficial lymphatic system and provides an indication of its functional status. This means changes to the superficial lymphatic system after soft tissue damage and reconstruction can be assessed.

Severe compound lower limb fractures are associated with extensive soft tissue damage resulting in disruption of lymphatic pathways. The extensive tissue damage often requires transposition of a flap consisting of muscle and/or soft tissue to close any existing defect. These interventions may also influence lymphatic regeneration and pathways. For these reasons an in-depth understanding of an individual’s local and general lymphatic architecture is valuable both in the understanding of underlying pathology and for the targeting and tailoring of treatment.

Materials and methods

A custom made near-infrared indocyanine green imaging system was designed and built with the Biomedical Engineering department at Flinders University. This system was tested and validated with in vitro and ex vivo experiments.

In addition, the system was used in experimentation with genetically modified mice (n=14, Gata2 heterozygous) with compromised lymphatic function versus wildtype mice (n=9).

Human research commenced following ethics approval at the Royal Adelaide Hospital. Patients who had a prior reconstructive surgery between 2009 and 2015 as a result of severe lower limb trauma were recruited from an existing database at its department of Plastic and Reconstructive Surgery, Royal Adelaide Hospital. At recruitment basic socio-demographic data was collected and a General Short Health questionnaire (SF-12), and the Lower Extremity Functioning Scale (LEFS) were self-administered. Baseline data at this time was also collected, including that relating to general and site specific fluids using Bio-impedance Spectroscopy (ImpediMed, Queensland) and Tissue Dielectric Constants (TDC) Moisture Meter (Delfin Technologies, Finland) respectively. To measure skin barrier function, Trans-Epidermal water loss was assessed, using a Vapometer (Delfin Technologies, Finland). Leg circumferences at 4 cm intervals were determined using a standard tape measure to determine segmental and overall lower limb volumes. To examine lymphatic function, 0.1ml ICG (25mg/5ml PULSION medical, Germany) was injected intra-dermally into two sites of the dorsum of the foot. In all cases the contra-lateral non injured leg acted as the control. All measurements were repeated at the 12-month follow-up with the patients’ consent.


The custom made imaging system showed fluorescence and worked effectively and reliably.

The Gata2 heterozygous mice (n=11) showed 61% faster uptake of lymph node fluorescence compared to wildtype mice (n=8). Lymphatic pulsation was detected and there was a faster pulsation in the wildtype (n=9, 0.20 beats per second) compared to the Gata2 heterozygous mice (n=12, 0.15 beats per second).

23 participants were recruited for the clinical study, 94% of the patients were male. The average age at presentation was 47 years with an average duration between the time of accident to the baseline observation of 38 months. Of the patients imaged in this study, 9 had free muscle flaps with a split thickness skin graft, 1 had a free fasciocutaneous flap, 1 full thickness skin graft, 6 a local fasciocutaneous flap and 1 a pedicled gastrocnemius flap. At baseline, the Lower Extremity Functioning Scale (LEFS) of the group was showed participants were on 56% of their normal functioning.

ICG imaging showed none of the free muscle flaps, or the free fasciocuteneous flap, had any functional superficial lymphatic vessels. The local fasciocutanous flaps and the skin graft patients all demonstrated impaired lymphatic vessel function and a dermal backflow pattern similar to that found in chronic lymphoedema. The local fasciocutaneous flaps all demonstrated lymphatic block at their scar tissue edge.

Generally, the uptake of the ICG was delayed in the affected leg compared to their respective control, indicating a reduced lymph transport capacity and flow.

Total water content and extracellular fluid content of the reconstructed leg was significantly higher versus the control leg. Circumference measurements showed a difference between the reconstructed leg and the control but this was not statistically significant.

At the 12 month follow up, 17 out of 23 were re-assessed, and similar results were recorded. Three patients in whom ICG showed superficial dermal back flow common in lymphoedema, were referred to receive further diagnostic testing including lymphoscintigraphy to explore their deeper lymphatic system functioning. This test showed all three to have damage or dysfunction to the deeper lymphatic collectors in the legs


ICG lymphography is a novel imaging technique that may provide information of the superficial lymphatic system pattern in and around the reconstructed area following lower limb trauma, and in small animal experimentation. Severe compound fractures and the associated soft tissue injury can result in significant lymphatic disruption and an increased risk for the development of chronic lymphoedema.

Keywords: Superficial lymphatics, lower limb trauma, lymphoedema, indocyanine green, lymphatic imaging

Subject: Surgery thesis, Medicine thesis

Thesis type: Doctor of Philosophy
Completed: 2016
School: School of Medicine
Supervisor: Professor Neil Piller