The present study demonstrates that the CEUS method can be used to visualise the anatomy and function of the superficial lymphatics in the upper limbs, offering a feasible radiological tool for the assessment of diseases and conditions affecting the normal lymphatic function such as in patients suffering from problems after breast cancer therapy. Visualisation of lymphatic pathways agrees with previous studies using microbubble contrast agent imaging for the sentinel lymph nodes and animal studies with cutaneous melanoma [18, 20, 22, 27]. The anatomical information on the lymphatic pathways is in agreement with findings from traditional lymphoscintigraphy and MR lymphangiography [8, 28].
CEUS microbubble technology
Previously, studies with CEUS have been mainly limited to the imaging of the SLNs in breast cancer and vulvar cancer following intradermal injection of contrast agent [22, 18,19,20]. To the best of our knowledge, the current study is the first to explore the potential of CEUS as a tool for mapping superficial lymphatics in humans. Although several other means for visualising and diagnosing possible lymphatic problems such as ICG and MR lymphangiography are nowadays available [9,10,11], there is a lack of a quick and cost-effective screening tool.
This study demonstrated in healthy volunteers that the CEUS procedure can be technically successful in 98.3% of upper limbs for visualising the pathways of the superficial lymphatics. The method proved to be quick since contrast agent enhancement in the axillar area was detected with a median time of 75 s after injection. These enhancement times are much faster than those necessary with ICG, which are about 30 min in the antecubital fossa [29]. The early enhancement time is evidently due to the small particle size of the microbubbles and the water-based solution of contrast agent. Due to the fast enhancement times, the contrast agent was seen in the superficial lymphatic vessels for a few minutes after the initial enhancement. Thus, these results could probably be achieved with the more commonly used contrast agent Sonovue® since it shares same kind of qualities as the Sonazoid® and the lifespan of the microbubbles exceeds the enhancement times shown in this study. Furthermore, it has been previously successfully used in the SLN studies to visualise lymphatic vessels [18,19,20, 30].
Factors related to intradermal injections
The intradermal injection technique is a procedure with a learning curve. In our study, a mean of 3.1 injections were needed in order to visualise the lymphatic collector vessels of both upper limbs. Successful intradermal injections were followed by fast lymphatic drainage and good visualisation of the contrast agent in lymphatic vessels (Table 2). However, successful injections could also result in slight-to-moderate visualisation of the lymphatics and longer enhancement times in elbow and axillar area. In some of the slower type B enhancements, the contrast agent was observed to be spreading into a larger area beneath the skin surface whereas more typically, intradermal injections produced a blister-like spot with a diameter of about 5 mm. Thus, the type B enhancements might be related to injections partly into dermis and partly into the upper subcutis and these injections could thus be defined as partially successful. In contrast, an indicator of a failed injection was the lack of resistance during the administration of contrast agent. If no enhancement was detected in the lymphatic vessels, it was evident that the injection had been totally subcutaneous.
In both type A (strong) and type B (moderate) enhancement patterns, the route of contrast agent was found to be identical and corresponded to the major pathways detailed in previous cadaver and ICG studies [7, 11]. Even the partially successful injections could thus also provide valuable information on the anatomy and function of the lymphatic drainage. The enhancement time results were registered from the first successful injection regardless of its enhancement type, leading to a relatively large variation in the enhancement times in our relatively small study population.
In our study, the alternative lymphatic pathways passing on the lateral aspect of the upper arm following the cephalic vein and connecting straight to supraclavicular lymph nodes were not visualised. Injections from secondary peripheral injection sites (Fig. 2), similar to those used by Suami et al. [11], only produced findings that the ipsilateral axilla drainage pathway was the major pathway of contrast agent; results are in agreement with those obtained with ICG fluorescence lymphography.
Initial findings in healthy volunteers
By dividing our group of volunteers according to gender, BMI, or into subjects young or older ages, we attempted to assess possible individual factors affecting the enhancement time of the contrast agent. If one considers gender-related differences, females displayed a 28% faster mean enhancement time than males in left antecubital fossa (18 s versus 23 s) with a slight statistically significant association. However, no clear reason for this phenomenon was identified and it might be affected by variables not included in the study such as arm length. Participants lay on the examination table and no muscle stress was induced voluntary that could affect the result. As massaging the injection site in the SLN studies to expedite the lymphatic flow [18, 20], the muscle contractions increase the lymph flow in skeletal muscle [6] and involuntary upper limb movement could potentially result in faster enhancement time. Otherwise no statistical differences were detected.
This microbubble contrast agent method proved to be feasible in mapping the superficial lymphatics of the upper limbs in healthy volunteers. The method needs to be further tested in patients with lymphatic disorders such as breast cancer-related lymphoedema associated with an abnormal distribution of lymph fluid into dermis. Studies planned for the future should help us to fully understand differences in CEUS lymphatic imaging between the upper limbs of healthy volunteers and patients with lymphoedema. Moreover, since lymphatic anatomy after breast cancer surgery may totally differ from normal lymphatic anatomy [31], benefits, and limitations of the CEUS for imaging the routes of abnormal lymphatic drainage in sites with dermal backflow need to be investigated. Furthermore, lymphatic CEUS studies in patients with breast cancer-related lymphoedema are therefore warranted since dermal backflow is considered as the most reliable indicator for lymphoedema [32] and US devices are widely available in healthcare units.