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Contrast-enhanced ultrasound: a new tool for imaging the superficial lymphatic vessels of the upper limb

Contrast-enhanced ultrasound: a new tool for imaging the superficial lymphatic vessels of the... Background: Despite the new lymphatic imaging methods, there is still a need for a straightforward method of detecting lymphatic abnormalities. Our goal was to investigate the feasibility of applying a contrast enhanced ultrasound (CEUS) procedure as a new approach for visualising the superficial lymphatic vessels of the upper limb. Methods: Thirty healthy volunteers were examined with CEUS after bilateral intradermal injection of Sonazoid® contrast agent in distal antebrachium. We registered factors affecting intradermal injections, imaging of the superficial lymphatic vessels and the enhancement time of contrast agent reaching the levels of elbow and axilla. Results: CEUS imaging of superficial lymphatic vessels was successful in 59 of 60 upper limbs (98.3%). Median [interquartile ranges] enhancement times of contrast agent to reach the elbow (right 18 s [11–25], left 15 s [12–25]) and axilla (right 77 s [33–118], left 66 s [42–115]) were equally fast. Successful intradermal injections were found to result in two types of contrast enhancement (strong or moderate), while the enhancement time depended on the type of the successful injection. No major differences in enhancement times were observed related to sex, body mass index, age, or side of the arm. Conclusions: The superficial lymphatic pathways of the upper limb can be visualised with CEUS imaging. Since enhancement time is dependent on the success of intradermal injections, one must pay attention to the injection technique. Further studies are needed to evaluate the method in patients with lymphatic function disorders such as breast cancer therapy related lymphoedema. Keywords: Contrast media, Injections (intradermal), Lymphatic vessels, Sonazoid, Ultrasonography Key points Background Despite the important role of the lymphatic system in Superficial lymphatic vessels of the upper limbs can human health and diseases, this system is still poorly be visualised with contrast-enhanced ultrasound. understood. After the pioneering work of Mascagni [1] Successful intradermal Sonazoid® injections were and Sappey [2] using a mercury injection method, recent followed by fast lymphatic drainage. studies with radiopaque lead oxide mixtures in cadavers Contrast enhancement in the axillary area was have led to a re-evaluation of the anatomical details of detected with a median time of 75 s after the the lymphatic pathways [3, 4]. Similar to the situation injection. with the vascular system, the anatomy of the lymphatic system consists of a complex network of small vessels. The lymphatic network is composed of the initial lym- * Correspondence: olli.lahtinen@kuh.fi Diagnostic Imaging Centre, Department of Clinical Radiology, Kuopio phatics or lymphatic capillaries, precollectors and col- University Hospital, Kuopio, Finland lecting vessels [5–7]. The lymphatic capillaries are blind- Institute of Clinical Medicine, Unit of Radiology, University of Eastern ended vessels containing a single layer of endothelial Finland, Kuopio, Finland © The Author(s) under exclusive licence to European Society of Radiology. 2022 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. Lahtinen et al. European Radiology Experimental (2022) 6:18 Page 2 of 9 cells. The diameter of the initial lymphatic vessels typic- Methods ally o ranges from 20 to 70 μm. Unlike the lymphatic ca- Patients and study design pillaries, precollectors and collecting vessels contain Approvals from our institutional review board, local ethics valves to prevent a backflow of lymph fluid. The walls of committee and Finnish Medicines Agency (FIMEA) were collecting vessels have smooth muscle cells and collagen obtained for this prospective single-centre study and all of fibres that maintain efficient lymph drainage [7]. How- the participants provided written informed consent. ever, unlike the vascular system the lymph drainage Thirty healthy volunteers were recruited to the study. through lymphatic vessels is not propelled by pulsatile Both upper limbs were independently evaluated in each blood circulation. subject. Exclusion criteria were (1) egg protein allergy; Traditionally, lymphoscintigraphy has been the (2) pregnancy; (3) breastfeeding; (4) prior axillary lymph- method of choice for diagnosing lymphatic disorders adenectomy. To allow us to evaluate age dependence, such as lymphoedema [8]. Recently, new lymphatic im- the volunteers were further divided into two groups: a aging methods have developed such as magnetic reson- younger group of persons less than 40 years of age and ance (MR) lymphangiography and indocyanine green an older group equal or greater than 40 years of age. (ICG) fluorescence imaging [9–11]. However, all these When studying the possible effect of body mass index imaging techniques are either time consuming, need to (BMI), the volunteers were divided into two groups: sub- expose the patient to radioactive tracer or are not cost jects with BMI < 25 and subjects with BMI > 25. effective on a large scale. Therefore, there is a need for a quick, easy and readily available tool with which to as- Upper limb US and CEUS sess common lymphatic abnormalities such as secondary The US procedures were performed by a single radiolo- lymphoedema after lymphadenectomies of breast cancer gist with 6 years of experience with CEUS. A Logiq TM surgery [12–14]. E9 US-device (General Electric Healthcare, Chicago, Ultrasound (US) might possess some of these advan- USA) with a broad spectrum ML6-15 (6–15 MHz) linear tages since the contrast agent used for contrast-enhanced array transducer. Participants were examined in supine ultrasound (CEUS) is a water-based solution like ICG position relaxed with no muscle stress. The applied which is known to travel faster in lymphatics than is the CEUS procedure was performed similar to US studies case with non-water based solutions. Ultrasound has been for the identification of SLNs [18, 20]. applied in imaging of the lymphatic system as is in the case of evaluation of lymph nodes (LN) [15]. In breast Microbubble contrast agent injection cancer-related lymphoedema, US can assess the response We hypothesised that the required enhancement times to physical therapy [16]. Recently, ultra-high frequency US for the evaluation of the whole upper limb would be lon- has been applied as an intraoperative planning tool in ger compared to those required in SLN studies. There- lymphatic microsurgery [17]. Additionally, the sentinel fore, the contrast agent Sonazoid® was selected for the lymph nodes (SLNs) and related afferent lymphatic vessels study as potentially being more durable compared to the have been successfully visualised with CEUS in breast and more commonly used Sonovue®. The contrast agent vulvar cancers [18–22]. powder, Sonazoid® was mixed with 2.0 mL of sterile sa- CEUS is one of the new US approaches using micro- line producing a microbubble solution with a mean par- bubbles such as those of perflubutane (Sonazoid®, GE ticle diameter of 2.6 μm. The Sonazoid® particles were Healthcare, Oslo, Norway) used in this study. Perflubu- injected intradermally with a 1.0-mL syringe and a 24- tane is a relatively new, second generation US contrast gauge needle laterally on the volar side of the distal ante- agent with some additional characteristics over the more brachium (Fig. 1). The injection was made with a low commonly used sulfur hexafluoride (Sonovue®, Bracco, angle, typically less than 5° from the skin. The tip of the Milan, Italy) such as its affinity for reticuloendothelial needle was positioned so that it could be seen through cells [23, 24]. Like sulfur hexafluoride, it is well tolerated the thin layer of epidermis. Injection was made as a fast and has no major contraindications or severe known ad- bolus. However, the injection rate depended on the re- verse effects when administered by intravenous or sub- sistance from the tissues. The timer was started after the dermal injections [25, 26]. whole volume was injected. The injected volume of the In the present study, our aim was to investigate the contrast agent solution was 0.2-mL for the whole study feasibility of applying CEUS to achieve a fast visualisa- population. If an enhancing lymphatic vessel was not de- tion of the superficial lymphatic vessels of the upper tected after injection or only minimal enhancement was limbs in healthy volunteers. In addition, we registered seen near the injection site in the CEUS image, the in- possible confounding factors related to the intradermal jection site was gently massaged for approximately 10 s. injection technique to be applied with the microbubble The injection was repeated up to three times at a nearby contrast agent. skin site within 1 cm from the first injection site if no Lahtinen et al. European Radiology Experimental (2022) 6:18 Page 3 of 9 Fig. 1 a Injection site in the distal antebrachium and intradermal injection technique. A small blister-like spot beneath the skin in addition to resistance to injection are probable indicators of a successful injection. Contrast agent (Sonazoid®, white arrows) can be seen in the superficial lymphatic vessel in b Fig. 2 The black X in a marks the primary injection site in the distal antebrachium. Additional injection sites are marked with a red X in a and b Lahtinen et al. European Radiology Experimental (2022) 6:18 Page 4 of 9 Fig. 3 The CEUS image in a correlates with a type A (strong enhancement) in the superficial lymphatic vessel, whereas the CEUS image in b corresponds to type B (moderate enhancement), which is clearly seen from the background but is clearly more faint than type A. White arrows mark the enhancing superficial lymphatic vessels additional enhancement was seen after the massage. In administration of the contrast agent. In addition, the addition, three other injection sites (Fig. 2) similar to participants were told to contact the main investigator those used by Suami et al. [11] were tested in each upper should any late onset symptoms develop within 72 h limb. Injected volumes of 0.1, 0.2, and 0.3 mL were after the contrast agent injection. tested in five volunteers after a successful injection to the primary site. Additional injections were made when Statistical analysis no residual enhancement was seen from the previous in- Variables are reported as median and interquartile range. jections. Continuous flash was used before the next in- Statistical analysis was performed with SPSS (Version jection to help to destroy the microbubbles. Thus, the 26.0, SPSS, Chicago, IL, USA). The Mann-Whitney U additional test volumes in the five volunteers were used test was used to calculate statistical significance between to test: (1) the feasibility with a lower dose; and (2) the different variables (dichotomised age, sex, BMI, whether a stronger enhancement type would follow with strength of the enhancement). Wilcoxon-related samples the larger dose. Besides feasibility assessment, no de- test was used to calculate inter-side differences between tailed evaluation of the different enhancement patterns right and left upper limbs of the appearance time of the or times from other injection sites was performed. contrast agent in antecubital fossa and axilla. Values of p The flow of contrast agent was followed using a standard lower than 0.05 were considered significant. low (< 0.10) mechanical index, as recommended by the manufacturer of Sonazoid®. The CEUS program allows the user to distinguish the echoes emitted by the microbubbles Results from tissue background and optimise the visualisation of Thirty healthy volunteers (16 males and 14 females, aged the contrast agent in the US image. The enhancement time 41 ± 11.2 years, mean ± standard deviation, range 27–69 from contrast agent injection to the detection of the years) were recruited between October 2020 and January contrast agent in antecubital fossa and axilla was registered 2021. Both upper limbs were evaluated in each subject. and major lymphatic pathways and the number of en- The BMI was 25.8 ± 4.8 (mean ± standard deviation, hanced lymphatic vessels were documented. The strength range 19.2–39.3). The right arm was dominant in 29 of the enhancement was divided into two categories by the persons. observer, depending on the visual appearance of the Intradermal contrast agent injections were successful contrast enhancement. Category A corresponded to strong in 59/60 (98.3%) of the upper limbs evaluated. A mean enhancement in the superficial lymphatic vessel while of 3.1 injections were needed to visualise the lymphatic category B referred to moderate enhancement (Fig. 3). collector vessels from upper arms combined. A clear in- dicator of a failed injection was the lack of resistance Follow-up during the administration of contrast agent. The number Participants were followed up for any possible acute on- of enhanced lymphatic collectors varied between 1 and 4 set of adverse effects for one hour after the with a mean of 1.5 vessels per upper limb that were Lahtinen et al. European Radiology Experimental (2022) 6:18 Page 5 of 9 Successful intradermal injections resulted into two types of contrast enhancement. In type A (n = 34), a high con- centration enhancement and a fast enhancement time of contrast agent in lymphatics were observed. Type B (n = 25) enhancements demonstrated less noticeable visual- isation of the lymphatics with a longer enhancement time in antecubital fossa and axillar area (Fig. 5). In some injections resulting in type B enhancements, the contrast agent was found to have spread into a larger area beneath the skin surface compared to the typical intradermal injections producing a blister-like spot. Thus, type A enhancement pattern could be charac- terised as being associated with a successful intradermal injection, and a type B enhancement pattern as related to a partially successful intradermal injection. Table 1 summarises the enhancement times of the contrast agent. There was no statistically significant difference in me- dian enhancement times to reach right antecubital fossa (18 s) and left antecubital fossa (15 s) (p = 0.863) or ax- illa (77 s and 66 s, respectively) (p= 0.581). BMI or youn- ger versus older age of volunteers had no significant effect on enhancement times. A slight difference was found between genders in the enhancement time in the left antecubital fossa, where females had 28% faster mean enhancement time compared to males (females 18 s versus males 23 s, p = 0.029). More detailed informa- tion is presented in Table 2. Additional test volumes were used in five volunteers. These demonstrated that in addition to a standard test dose of 0.2 mL, lymphatic vessels could also be visua- lised with doses of 0.1 mL and 0.3 mL. Furthermore, in- jections of contrast agent into one additional volar site and into two sites in dorsal aspects on back of the hand were also feasible to visualise the superficial lymphatic routes. The lymph drainage from these additional sites followed the same pathway as with the main injection site on the volar aspect of the distal antebrachium. The alternative lymphatic pathways passing on the lateral as- pect of the upper arm following the cephalic vein and Fig. 4 The red X marks the injection site of the contrast agent. Blue dots demonstrate the location of the enhancing lymph nodes in the connecting straight to the supraclavicular lymph nodes antecubital fossa and axilla. The red line illustrates roughly the were not visualised in this study. In two subjects, an en- enhancing superficial lymphatic route seen in all of the hancing lymph node following the main pathway was successful injections found in the medial aspect of the elbow (Fig. 4). No adverse effects were reported related to the intra- dermal injections or CEUS microbubble contrast agent. located in parallel. In all volunteers, the contrast agent followed the same lymphatic pathway (Fig. 4). Discussion The enhancement time of the contrast agent depended The present study demonstrates that the CEUS method on the success of intradermal injections. Typically, a suc- can be used to visualise the anatomy and function of the cessful intradermal injection produced a blister-like spot superficial lymphatics in the upper limbs, offering a feas- with a diameter of about 5 mm. None or only minimal ible radiological tool for the assessment of diseases and visualisation of the lymphatics was registered if the injec- conditions affecting the normal lymphatic function such tion of contrast agent had occurred subcutaneously. as in patients suffering from problems after breast Lahtinen et al. European Radiology Experimental (2022) 6:18 Page 6 of 9 Fig. 5 Enhancement times in antecubital fossa (a, c) and axilla (b, d) displayed variance according to the two US enhancement categories (type A = strong, type B = moderate) in the right and left arms 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 Table 1 Enhancement times in the antecubital fossa and axilla agreement with findings from traditional lymphoscinti- after intradermal injections graphy and MR lymphangiography [8, 28]. Assessment site Enhancement time (s) Median (interquartile range) CEUS microbubble technology Right antecubital fossa 18 (11–25) Previously, studies with CEUS have been mainly limited Left antecubital fossa 15 (12–25) to the imaging of the SLNs in breast cancer and vulvar Right axilla 77 (33–118) cancer following intradermal injection of contrast agent Left axilla 66 (42–115) [22, 18–20]. To the best of our knowledge, the current Lahtinen et al. European Radiology Experimental (2022) 6:18 Page 7 of 9 Table 2 Effect of different parameters on the enhancement time after successful intradermal injections Enhancement time (s) Median (interquartile range) Parameter Number Antecubital fossa Axilla Right Left Right Left Sex Males 16 15 (10–31) 20 (13–25) 57 (31–120) 98 (52–133) Females 14 19 (12–25) 12 (9–21) 81 (42–116) 49 (31–96) p 0.455 0.029 0.583 0.070 Age (years) < 40 18 18 (11–34) 19 (11–29) 77 (33–123) 92 (49–147) > 40 12 18 (11–25) 15 (12–24) 77 (32–109) 50 (37–95) p 0.929 0.484 0.825 0.086 Body mass index < 25 16 19 (13–33) 17 (11–23) 80 (40–123) 69 (39–133) > 25 14 14 (11–25) 14 (12–35) 57 (28–99) 63 (44–112) p 0.256 0.546 0.111 0.884 Right+ left Enhancement type A 15 + 19 = 34 12 (9–20) 13 (10–19) 36 (29–49) 50 (32–70) B 14 + 11 = 25 25 (18–46) 25 (20–34) 118 (88–134) 126 (95–191) p 0.001 0.007 0.000 0.000 Enhancement type A indicates a successful intradermal injection with strong visual enhancement, whereas type B refers to a partially successful injection with moderate enhancement. Enhancement times in the table represent median enhancement times at the antecubital fossa and axilla study is the first to explore the potential of CEUS as a vessels of both upper limbs. Successful intradermal in- tool for mapping superficial lymphatics in humans. Al- jections were followed by fast lymphatic drainage and though several other means for visualising and diagnos- good visualisation of the contrast agent in lymphatic ves- ing possible lymphatic problems such as ICG and MR sels (Table 2). However, successful injections could also lymphangiography are nowadays available [9–11], there result in slight-to-moderate visualisation of the lym- is a lack of a quick and cost-effective screening tool. phatics and longer enhancement times in elbow and ax- This study demonstrated in healthy volunteers that the illar area. In some of the slower type B enhancements, CEUS procedure can be technically successful in 98.3% the contrast agent was observed to be spreading into a of upper limbs for visualising the pathways of the super- larger area beneath the skin surface whereas more typic- ficial lymphatics. The method proved to be quick since ally, intradermal injections produced a blister-like spot contrast agent enhancement in the axillar area was de- with a diameter of about 5 mm. Thus, the type B tected with a median time of 75 s after injection. These enhancements might be related to injections partly enhancement times are much faster than those necessary into dermis and partly into the upper subcutis and with ICG, which are about 30 min in the antecubital these injections could thus be defined as partially fossa [29]. The early enhancement time is evidently due successful. In contrast, an indicator of a failed injec- to the small particle size of the microbubbles and the tion was the lack of resistance during the adminis- water-based solution of contrast agent. Due to the fast tration of contrast agent. If no enhancement was enhancement times, the contrast agent was seen in the detected in the lymphatic vessels, it was evident that superficial lymphatic vessels for a few minutes after the the injection had been totally subcutaneous. initial enhancement. Thus, these results could probably In both type A (strong) and type B (moderate) en- be achieved with the more commonly used contrast hancement patterns, the route of contrast agent was agent Sonovue® since it shares same kind of qualities as foundtobeidentical andcorrespondedtothe major the Sonazoid® and the lifespan of the microbubbles ex- pathways detailed in previous cadaver and ICG stud- ceeds the enhancement times shown in this study. Fur- ies [7, 11]. Even the partially successful injections thermore, it has been previously successfully used in the could thus also provide valuable information on the SLN studies to visualise lymphatic vessels [18–20, 30]. anatomy and function of the lymphatic drainage. The enhancement time results were registered from Factors related to intradermal injections the first successful injection regardless of its en- The intradermal injection technique is a procedure with hancement type, leading to a relatively large vari- a learning curve. In our study, a mean of 3.1 injections ation in the enhancement times in our relatively were needed in order to visualise the lymphatic collector small study population. Lahtinen et al. European Radiology Experimental (2022) 6:18 Page 8 of 9 In our study, the alternative lymphatic pathways as whether the intradermal injection was successful, passing on the lateral aspect of the upper arm fol- leading to a type A (strong) enhancement or partially lowing the cephalic vein and connecting straight to successful leading to a relatively large number of type B supraclavicular lymph nodes were not visualised. In- (moderate) enhancements. Classification into these two jections from secondary peripheral injection sites types was subjective although the difference could be (Fig. 2), similar to those used by Suami et al. [11], seen on the live display. The use of time intensity curve only produced findings that the ipsilateral axilla analysis, not available for this study, could provide more drainage pathway was the major pathway of contrast quantitative results of these types in the future. agent; results are in agreement with those obtained Another limitation is the lack of multiple observers, with ICG fluorescence lymphography. also adding subjectivity to our results. However, a simi- lar injection technique has been used in SLN studies and Initial findings in healthy volunteers has shown to be reproducible both in patients with By dividing our group of volunteers according to gender, breast cancer and vulvar cancer [20, 21]. Since enhance- BMI, or into subjects young or older ages, we attempted ment time is dependent on the success of intradermal to assess possible individual factors affecting the en- injections, the injection technique demands special care hancement time of the contrast agent. If one considers to avoid unsuccessful injection. A reproducible injection gender-related differences, females displayed a 28% fas- technique is crucial if one is to differentiate real lymph- ter mean enhancement time than males in left antecubi- atic disorders from injection-induced effects. Further tal fossa (18 s versus 23 s) with a slight statistically studies investigating the CEUS technique in the longer significant association. However, no clear reason for this range of the whole upper limb are warranted to fully phenomenon was identified and it might be affected by clarify the differences between normal and abnormal variables not included in the study such as arm length. lymphatic anatomy. Participants lay on the examination table and no muscle stress was induced voluntary that could affect the result. Conclusions As massaging the injection site in the SLN studies to ex- From this preliminary study, we conclude that the CEUS pedite the lymphatic flow [18, 20], the muscle contrac- method following intradermal injections was able to tions increase the lymph flow in skeletal muscle [6] and identify the superficial lymphatic pathways in the upper involuntary upper limb movement could potentially re- arms of healthy volunteers. CEUS represents a potential sult in faster enhancement time. Otherwise no statistical minimally invasive tool with which to assess the kinetics differences were detected. of lymph fluid and allow the imaging of abnormal This microbubble contrast agent method proved to be lymphatic anatomy. feasible in mapping the superficial lymphatics of the Abbreviations upper limbs in healthy volunteers. The method needs to BMI: Body mass index; CEUS: Contrast-enhanced ultrasound; ICG: Indocyanine be further tested in patients with lymphatic disorders green; MR: Magnetic resonance; SLN: Sentinel lymph node; US: Ultrasound such as breast cancer-related lymphoedema associated Authors’ contributions with an abnormal distribution of lymph fluid into der- OL was the main investigator. He analysed, interpreted the data, and was the mis. Studies planned for the future should help us to writer of the manuscript. RV and SR contributed to writing of the manuscript. fully understand differences in CEUS lymphatic imaging All authors read and approved the final manuscript. between the upper limbs of healthy volunteers and pa- Funding tients with lymphoedema. Moreover, since lymphatic This study has received funding from the Paavo Koistinen foundation (grant anatomy after breast cancer surgery may totally differ number 20200021) and Kuopio University Hospital Science Foundation (grant). from normal lymphatic anatomy [31], benefits, and limi- tations of the CEUS for imaging the routes of abnormal Availability of data and materials lymphatic drainage in sites with dermal backflow need The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request. to be investigated. Furthermore, lymphatic CEUS studies in patients with breast cancer-related lymphoedema are Declarations therefore warranted since dermal backflow is considered Ethics approval and consent to participate as the most reliable indicator for lymphoedema [32] and Approvals from our institutional review board, local ethics committee US devices are widely available in healthcare units. (Kuopio University Hospital) and Finnish Medicines Agency (FIMEA) were obtained for this prospective single-center study and all of the participants provided written informed consent. Study limitations The present study has several limitations. Enhancement Consent for publication times were registered quantitatively, but the division into All the participants in the study have given written consent to use the data two different enhancement types was done subjectively collected in the study as a part of the manuscript. Lahtinen et al. European Radiology Experimental (2022) 6:18 Page 9 of 9 Competing interests 22. Wang Y, Zhou W, Li C, et al (2017) Variation of sentinel lymphatic channels The authors declare that they have no competing interests. (SLCs) and sentinel lymph nodes (SLNs) assessed by contrast-enhanced ultrasound (CEUS) in breast cancer patients. World J Surg Oncol 15:127. Received: 17 December 2021 Accepted: 9 March 2022 https://doi.org/10.1186/s12957-017-1195-3 23. 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Contrast-enhanced ultrasound: a new tool for imaging the superficial lymphatic vessels of the upper limb

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Abstract

Background: Despite the new lymphatic imaging methods, there is still a need for a straightforward method of detecting lymphatic abnormalities. Our goal was to investigate the feasibility of applying a contrast enhanced ultrasound (CEUS) procedure as a new approach for visualising the superficial lymphatic vessels of the upper limb. Methods: Thirty healthy volunteers were examined with CEUS after bilateral intradermal injection of Sonazoid® contrast agent in distal antebrachium. We registered factors affecting intradermal injections, imaging of the superficial lymphatic vessels and the enhancement time of contrast agent reaching the levels of elbow and axilla. Results: CEUS imaging of superficial lymphatic vessels was successful in 59 of 60 upper limbs (98.3%). Median [interquartile ranges] enhancement times of contrast agent to reach the elbow (right 18 s [11–25], left 15 s [12–25]) and axilla (right 77 s [33–118], left 66 s [42–115]) were equally fast. Successful intradermal injections were found to result in two types of contrast enhancement (strong or moderate), while the enhancement time depended on the type of the successful injection. No major differences in enhancement times were observed related to sex, body mass index, age, or side of the arm. Conclusions: The superficial lymphatic pathways of the upper limb can be visualised with CEUS imaging. Since enhancement time is dependent on the success of intradermal injections, one must pay attention to the injection technique. Further studies are needed to evaluate the method in patients with lymphatic function disorders such as breast cancer therapy related lymphoedema. Keywords: Contrast media, Injections (intradermal), Lymphatic vessels, Sonazoid, Ultrasonography Key points Background Despite the important role of the lymphatic system in Superficial lymphatic vessels of the upper limbs can human health and diseases, this system is still poorly be visualised with contrast-enhanced ultrasound. understood. After the pioneering work of Mascagni [1] Successful intradermal Sonazoid® injections were and Sappey [2] using a mercury injection method, recent followed by fast lymphatic drainage. studies with radiopaque lead oxide mixtures in cadavers Contrast enhancement in the axillary area was have led to a re-evaluation of the anatomical details of detected with a median time of 75 s after the the lymphatic pathways [3, 4]. Similar to the situation injection. with the vascular system, the anatomy of the lymphatic system consists of a complex network of small vessels. The lymphatic network is composed of the initial lym- * Correspondence: olli.lahtinen@kuh.fi Diagnostic Imaging Centre, Department of Clinical Radiology, Kuopio phatics or lymphatic capillaries, precollectors and col- University Hospital, Kuopio, Finland lecting vessels [5–7]. The lymphatic capillaries are blind- Institute of Clinical Medicine, Unit of Radiology, University of Eastern ended vessels containing a single layer of endothelial Finland, Kuopio, Finland © The Author(s) under exclusive licence to European Society of Radiology. 2022 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. Lahtinen et al. European Radiology Experimental (2022) 6:18 Page 2 of 9 cells. The diameter of the initial lymphatic vessels typic- Methods ally o ranges from 20 to 70 μm. Unlike the lymphatic ca- Patients and study design pillaries, precollectors and collecting vessels contain Approvals from our institutional review board, local ethics valves to prevent a backflow of lymph fluid. The walls of committee and Finnish Medicines Agency (FIMEA) were collecting vessels have smooth muscle cells and collagen obtained for this prospective single-centre study and all of fibres that maintain efficient lymph drainage [7]. How- the participants provided written informed consent. ever, unlike the vascular system the lymph drainage Thirty healthy volunteers were recruited to the study. through lymphatic vessels is not propelled by pulsatile Both upper limbs were independently evaluated in each blood circulation. subject. Exclusion criteria were (1) egg protein allergy; Traditionally, lymphoscintigraphy has been the (2) pregnancy; (3) breastfeeding; (4) prior axillary lymph- method of choice for diagnosing lymphatic disorders adenectomy. To allow us to evaluate age dependence, such as lymphoedema [8]. Recently, new lymphatic im- the volunteers were further divided into two groups: a aging methods have developed such as magnetic reson- younger group of persons less than 40 years of age and ance (MR) lymphangiography and indocyanine green an older group equal or greater than 40 years of age. (ICG) fluorescence imaging [9–11]. However, all these When studying the possible effect of body mass index imaging techniques are either time consuming, need to (BMI), the volunteers were divided into two groups: sub- expose the patient to radioactive tracer or are not cost jects with BMI < 25 and subjects with BMI > 25. effective on a large scale. Therefore, there is a need for a quick, easy and readily available tool with which to as- Upper limb US and CEUS sess common lymphatic abnormalities such as secondary The US procedures were performed by a single radiolo- lymphoedema after lymphadenectomies of breast cancer gist with 6 years of experience with CEUS. A Logiq TM surgery [12–14]. E9 US-device (General Electric Healthcare, Chicago, Ultrasound (US) might possess some of these advan- USA) with a broad spectrum ML6-15 (6–15 MHz) linear tages since the contrast agent used for contrast-enhanced array transducer. Participants were examined in supine ultrasound (CEUS) is a water-based solution like ICG position relaxed with no muscle stress. The applied which is known to travel faster in lymphatics than is the CEUS procedure was performed similar to US studies case with non-water based solutions. Ultrasound has been for the identification of SLNs [18, 20]. applied in imaging of the lymphatic system as is in the case of evaluation of lymph nodes (LN) [15]. In breast Microbubble contrast agent injection cancer-related lymphoedema, US can assess the response We hypothesised that the required enhancement times to physical therapy [16]. Recently, ultra-high frequency US for the evaluation of the whole upper limb would be lon- has been applied as an intraoperative planning tool in ger compared to those required in SLN studies. There- lymphatic microsurgery [17]. Additionally, the sentinel fore, the contrast agent Sonazoid® was selected for the lymph nodes (SLNs) and related afferent lymphatic vessels study as potentially being more durable compared to the have been successfully visualised with CEUS in breast and more commonly used Sonovue®. The contrast agent vulvar cancers [18–22]. powder, Sonazoid® was mixed with 2.0 mL of sterile sa- CEUS is one of the new US approaches using micro- line producing a microbubble solution with a mean par- bubbles such as those of perflubutane (Sonazoid®, GE ticle diameter of 2.6 μm. The Sonazoid® particles were Healthcare, Oslo, Norway) used in this study. Perflubu- injected intradermally with a 1.0-mL syringe and a 24- tane is a relatively new, second generation US contrast gauge needle laterally on the volar side of the distal ante- agent with some additional characteristics over the more brachium (Fig. 1). The injection was made with a low commonly used sulfur hexafluoride (Sonovue®, Bracco, angle, typically less than 5° from the skin. The tip of the Milan, Italy) such as its affinity for reticuloendothelial needle was positioned so that it could be seen through cells [23, 24]. Like sulfur hexafluoride, it is well tolerated the thin layer of epidermis. Injection was made as a fast and has no major contraindications or severe known ad- bolus. However, the injection rate depended on the re- verse effects when administered by intravenous or sub- sistance from the tissues. The timer was started after the dermal injections [25, 26]. whole volume was injected. The injected volume of the In the present study, our aim was to investigate the contrast agent solution was 0.2-mL for the whole study feasibility of applying CEUS to achieve a fast visualisa- population. If an enhancing lymphatic vessel was not de- tion of the superficial lymphatic vessels of the upper tected after injection or only minimal enhancement was limbs in healthy volunteers. In addition, we registered seen near the injection site in the CEUS image, the in- possible confounding factors related to the intradermal jection site was gently massaged for approximately 10 s. injection technique to be applied with the microbubble The injection was repeated up to three times at a nearby contrast agent. skin site within 1 cm from the first injection site if no Lahtinen et al. European Radiology Experimental (2022) 6:18 Page 3 of 9 Fig. 1 a Injection site in the distal antebrachium and intradermal injection technique. A small blister-like spot beneath the skin in addition to resistance to injection are probable indicators of a successful injection. Contrast agent (Sonazoid®, white arrows) can be seen in the superficial lymphatic vessel in b Fig. 2 The black X in a marks the primary injection site in the distal antebrachium. Additional injection sites are marked with a red X in a and b Lahtinen et al. European Radiology Experimental (2022) 6:18 Page 4 of 9 Fig. 3 The CEUS image in a correlates with a type A (strong enhancement) in the superficial lymphatic vessel, whereas the CEUS image in b corresponds to type B (moderate enhancement), which is clearly seen from the background but is clearly more faint than type A. White arrows mark the enhancing superficial lymphatic vessels additional enhancement was seen after the massage. In administration of the contrast agent. In addition, the addition, three other injection sites (Fig. 2) similar to participants were told to contact the main investigator those used by Suami et al. [11] were tested in each upper should any late onset symptoms develop within 72 h limb. Injected volumes of 0.1, 0.2, and 0.3 mL were after the contrast agent injection. tested in five volunteers after a successful injection to the primary site. Additional injections were made when Statistical analysis no residual enhancement was seen from the previous in- Variables are reported as median and interquartile range. jections. Continuous flash was used before the next in- Statistical analysis was performed with SPSS (Version jection to help to destroy the microbubbles. Thus, the 26.0, SPSS, Chicago, IL, USA). The Mann-Whitney U additional test volumes in the five volunteers were used test was used to calculate statistical significance between to test: (1) the feasibility with a lower dose; and (2) the different variables (dichotomised age, sex, BMI, whether a stronger enhancement type would follow with strength of the enhancement). Wilcoxon-related samples the larger dose. Besides feasibility assessment, no de- test was used to calculate inter-side differences between tailed evaluation of the different enhancement patterns right and left upper limbs of the appearance time of the or times from other injection sites was performed. contrast agent in antecubital fossa and axilla. Values of p The flow of contrast agent was followed using a standard lower than 0.05 were considered significant. low (< 0.10) mechanical index, as recommended by the manufacturer of Sonazoid®. The CEUS program allows the user to distinguish the echoes emitted by the microbubbles Results from tissue background and optimise the visualisation of Thirty healthy volunteers (16 males and 14 females, aged the contrast agent in the US image. The enhancement time 41 ± 11.2 years, mean ± standard deviation, range 27–69 from contrast agent injection to the detection of the years) were recruited between October 2020 and January contrast agent in antecubital fossa and axilla was registered 2021. Both upper limbs were evaluated in each subject. and major lymphatic pathways and the number of en- The BMI was 25.8 ± 4.8 (mean ± standard deviation, hanced lymphatic vessels were documented. The strength range 19.2–39.3). The right arm was dominant in 29 of the enhancement was divided into two categories by the persons. observer, depending on the visual appearance of the Intradermal contrast agent injections were successful contrast enhancement. Category A corresponded to strong in 59/60 (98.3%) of the upper limbs evaluated. A mean enhancement in the superficial lymphatic vessel while of 3.1 injections were needed to visualise the lymphatic category B referred to moderate enhancement (Fig. 3). collector vessels from upper arms combined. A clear in- dicator of a failed injection was the lack of resistance Follow-up during the administration of contrast agent. The number Participants were followed up for any possible acute on- of enhanced lymphatic collectors varied between 1 and 4 set of adverse effects for one hour after the with a mean of 1.5 vessels per upper limb that were Lahtinen et al. European Radiology Experimental (2022) 6:18 Page 5 of 9 Successful intradermal injections resulted into two types of contrast enhancement. In type A (n = 34), a high con- centration enhancement and a fast enhancement time of contrast agent in lymphatics were observed. Type B (n = 25) enhancements demonstrated less noticeable visual- isation of the lymphatics with a longer enhancement time in antecubital fossa and axillar area (Fig. 5). In some injections resulting in type B enhancements, the contrast agent was found to have spread into a larger area beneath the skin surface compared to the typical intradermal injections producing a blister-like spot. Thus, type A enhancement pattern could be charac- terised as being associated with a successful intradermal injection, and a type B enhancement pattern as related to a partially successful intradermal injection. Table 1 summarises the enhancement times of the contrast agent. There was no statistically significant difference in me- dian enhancement times to reach right antecubital fossa (18 s) and left antecubital fossa (15 s) (p = 0.863) or ax- illa (77 s and 66 s, respectively) (p= 0.581). BMI or youn- ger versus older age of volunteers had no significant effect on enhancement times. A slight difference was found between genders in the enhancement time in the left antecubital fossa, where females had 28% faster mean enhancement time compared to males (females 18 s versus males 23 s, p = 0.029). More detailed informa- tion is presented in Table 2. Additional test volumes were used in five volunteers. These demonstrated that in addition to a standard test dose of 0.2 mL, lymphatic vessels could also be visua- lised with doses of 0.1 mL and 0.3 mL. Furthermore, in- jections of contrast agent into one additional volar site and into two sites in dorsal aspects on back of the hand were also feasible to visualise the superficial lymphatic routes. The lymph drainage from these additional sites followed the same pathway as with the main injection site on the volar aspect of the distal antebrachium. The alternative lymphatic pathways passing on the lateral as- pect of the upper arm following the cephalic vein and Fig. 4 The red X marks the injection site of the contrast agent. Blue dots demonstrate the location of the enhancing lymph nodes in the connecting straight to the supraclavicular lymph nodes antecubital fossa and axilla. The red line illustrates roughly the were not visualised in this study. In two subjects, an en- enhancing superficial lymphatic route seen in all of the hancing lymph node following the main pathway was successful injections found in the medial aspect of the elbow (Fig. 4). No adverse effects were reported related to the intra- dermal injections or CEUS microbubble contrast agent. located in parallel. In all volunteers, the contrast agent followed the same lymphatic pathway (Fig. 4). Discussion The enhancement time of the contrast agent depended The present study demonstrates that the CEUS method on the success of intradermal injections. Typically, a suc- can be used to visualise the anatomy and function of the cessful intradermal injection produced a blister-like spot superficial lymphatics in the upper limbs, offering a feas- with a diameter of about 5 mm. None or only minimal ible radiological tool for the assessment of diseases and visualisation of the lymphatics was registered if the injec- conditions affecting the normal lymphatic function such tion of contrast agent had occurred subcutaneously. as in patients suffering from problems after breast Lahtinen et al. European Radiology Experimental (2022) 6:18 Page 6 of 9 Fig. 5 Enhancement times in antecubital fossa (a, c) and axilla (b, d) displayed variance according to the two US enhancement categories (type A = strong, type B = moderate) in the right and left arms 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 Table 1 Enhancement times in the antecubital fossa and axilla agreement with findings from traditional lymphoscinti- after intradermal injections graphy and MR lymphangiography [8, 28]. Assessment site Enhancement time (s) Median (interquartile range) CEUS microbubble technology Right antecubital fossa 18 (11–25) Previously, studies with CEUS have been mainly limited Left antecubital fossa 15 (12–25) to the imaging of the SLNs in breast cancer and vulvar Right axilla 77 (33–118) cancer following intradermal injection of contrast agent Left axilla 66 (42–115) [22, 18–20]. To the best of our knowledge, the current Lahtinen et al. European Radiology Experimental (2022) 6:18 Page 7 of 9 Table 2 Effect of different parameters on the enhancement time after successful intradermal injections Enhancement time (s) Median (interquartile range) Parameter Number Antecubital fossa Axilla Right Left Right Left Sex Males 16 15 (10–31) 20 (13–25) 57 (31–120) 98 (52–133) Females 14 19 (12–25) 12 (9–21) 81 (42–116) 49 (31–96) p 0.455 0.029 0.583 0.070 Age (years) < 40 18 18 (11–34) 19 (11–29) 77 (33–123) 92 (49–147) > 40 12 18 (11–25) 15 (12–24) 77 (32–109) 50 (37–95) p 0.929 0.484 0.825 0.086 Body mass index < 25 16 19 (13–33) 17 (11–23) 80 (40–123) 69 (39–133) > 25 14 14 (11–25) 14 (12–35) 57 (28–99) 63 (44–112) p 0.256 0.546 0.111 0.884 Right+ left Enhancement type A 15 + 19 = 34 12 (9–20) 13 (10–19) 36 (29–49) 50 (32–70) B 14 + 11 = 25 25 (18–46) 25 (20–34) 118 (88–134) 126 (95–191) p 0.001 0.007 0.000 0.000 Enhancement type A indicates a successful intradermal injection with strong visual enhancement, whereas type B refers to a partially successful injection with moderate enhancement. Enhancement times in the table represent median enhancement times at the antecubital fossa and axilla study is the first to explore the potential of CEUS as a vessels of both upper limbs. Successful intradermal in- tool for mapping superficial lymphatics in humans. Al- jections were followed by fast lymphatic drainage and though several other means for visualising and diagnos- good visualisation of the contrast agent in lymphatic ves- ing possible lymphatic problems such as ICG and MR sels (Table 2). However, successful injections could also lymphangiography are nowadays available [9–11], there result in slight-to-moderate visualisation of the lym- is a lack of a quick and cost-effective screening tool. phatics and longer enhancement times in elbow and ax- This study demonstrated in healthy volunteers that the illar area. In some of the slower type B enhancements, CEUS procedure can be technically successful in 98.3% the contrast agent was observed to be spreading into a of upper limbs for visualising the pathways of the super- larger area beneath the skin surface whereas more typic- ficial lymphatics. The method proved to be quick since ally, intradermal injections produced a blister-like spot contrast agent enhancement in the axillar area was de- with a diameter of about 5 mm. Thus, the type B tected with a median time of 75 s after injection. These enhancements might be related to injections partly enhancement times are much faster than those necessary into dermis and partly into the upper subcutis and with ICG, which are about 30 min in the antecubital these injections could thus be defined as partially fossa [29]. The early enhancement time is evidently due successful. In contrast, an indicator of a failed injec- to the small particle size of the microbubbles and the tion was the lack of resistance during the adminis- water-based solution of contrast agent. Due to the fast tration of contrast agent. If no enhancement was enhancement times, the contrast agent was seen in the detected in the lymphatic vessels, it was evident that superficial lymphatic vessels for a few minutes after the the injection had been totally subcutaneous. initial enhancement. Thus, these results could probably In both type A (strong) and type B (moderate) en- be achieved with the more commonly used contrast hancement patterns, the route of contrast agent was agent Sonovue® since it shares same kind of qualities as foundtobeidentical andcorrespondedtothe major the Sonazoid® and the lifespan of the microbubbles ex- pathways detailed in previous cadaver and ICG stud- ceeds the enhancement times shown in this study. Fur- ies [7, 11]. Even the partially successful injections thermore, it has been previously successfully used in the could thus also provide valuable information on the SLN studies to visualise lymphatic vessels [18–20, 30]. anatomy and function of the lymphatic drainage. The enhancement time results were registered from Factors related to intradermal injections the first successful injection regardless of its en- The intradermal injection technique is a procedure with hancement type, leading to a relatively large vari- a learning curve. In our study, a mean of 3.1 injections ation in the enhancement times in our relatively were needed in order to visualise the lymphatic collector small study population. Lahtinen et al. European Radiology Experimental (2022) 6:18 Page 8 of 9 In our study, the alternative lymphatic pathways as whether the intradermal injection was successful, passing on the lateral aspect of the upper arm fol- leading to a type A (strong) enhancement or partially lowing the cephalic vein and connecting straight to successful leading to a relatively large number of type B supraclavicular lymph nodes were not visualised. In- (moderate) enhancements. Classification into these two jections from secondary peripheral injection sites types was subjective although the difference could be (Fig. 2), similar to those used by Suami et al. [11], seen on the live display. The use of time intensity curve only produced findings that the ipsilateral axilla analysis, not available for this study, could provide more drainage pathway was the major pathway of contrast quantitative results of these types in the future. agent; results are in agreement with those obtained Another limitation is the lack of multiple observers, with ICG fluorescence lymphography. also adding subjectivity to our results. However, a simi- lar injection technique has been used in SLN studies and Initial findings in healthy volunteers has shown to be reproducible both in patients with By dividing our group of volunteers according to gender, breast cancer and vulvar cancer [20, 21]. Since enhance- BMI, or into subjects young or older ages, we attempted ment time is dependent on the success of intradermal to assess possible individual factors affecting the en- injections, the injection technique demands special care hancement time of the contrast agent. If one considers to avoid unsuccessful injection. A reproducible injection gender-related differences, females displayed a 28% fas- technique is crucial if one is to differentiate real lymph- ter mean enhancement time than males in left antecubi- atic disorders from injection-induced effects. Further tal fossa (18 s versus 23 s) with a slight statistically studies investigating the CEUS technique in the longer significant association. However, no clear reason for this range of the whole upper limb are warranted to fully phenomenon was identified and it might be affected by clarify the differences between normal and abnormal variables not included in the study such as arm length. lymphatic anatomy. Participants lay on the examination table and no muscle stress was induced voluntary that could affect the result. Conclusions As massaging the injection site in the SLN studies to ex- From this preliminary study, we conclude that the CEUS pedite the lymphatic flow [18, 20], the muscle contrac- method following intradermal injections was able to tions increase the lymph flow in skeletal muscle [6] and identify the superficial lymphatic pathways in the upper involuntary upper limb movement could potentially re- arms of healthy volunteers. CEUS represents a potential sult in faster enhancement time. Otherwise no statistical minimally invasive tool with which to assess the kinetics differences were detected. of lymph fluid and allow the imaging of abnormal This microbubble contrast agent method proved to be lymphatic anatomy. feasible in mapping the superficial lymphatics of the Abbreviations upper limbs in healthy volunteers. The method needs to BMI: Body mass index; CEUS: Contrast-enhanced ultrasound; ICG: Indocyanine be further tested in patients with lymphatic disorders green; MR: Magnetic resonance; SLN: Sentinel lymph node; US: Ultrasound such as breast cancer-related lymphoedema associated Authors’ contributions with an abnormal distribution of lymph fluid into der- OL was the main investigator. He analysed, interpreted the data, and was the mis. Studies planned for the future should help us to writer of the manuscript. RV and SR contributed to writing of the manuscript. fully understand differences in CEUS lymphatic imaging All authors read and approved the final manuscript. between the upper limbs of healthy volunteers and pa- Funding tients with lymphoedema. Moreover, since lymphatic This study has received funding from the Paavo Koistinen foundation (grant anatomy after breast cancer surgery may totally differ number 20200021) and Kuopio University Hospital Science Foundation (grant). from normal lymphatic anatomy [31], benefits, and limi- tations of the CEUS for imaging the routes of abnormal Availability of data and materials lymphatic drainage in sites with dermal backflow need The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request. to be investigated. Furthermore, lymphatic CEUS studies in patients with breast cancer-related lymphoedema are Declarations therefore warranted since dermal backflow is considered Ethics approval and consent to participate as the most reliable indicator for lymphoedema [32] and Approvals from our institutional review board, local ethics committee US devices are widely available in healthcare units. (Kuopio University Hospital) and Finnish Medicines Agency (FIMEA) were obtained for this prospective single-center study and all of the participants provided written informed consent. Study limitations The present study has several limitations. Enhancement Consent for publication times were registered quantitatively, but the division into All the participants in the study have given written consent to use the data two different enhancement types was done subjectively collected in the study as a part of the manuscript. Lahtinen et al. European Radiology Experimental (2022) 6:18 Page 9 of 9 Competing interests 22. Wang Y, Zhou W, Li C, et al (2017) Variation of sentinel lymphatic channels The authors declare that they have no competing interests. (SLCs) and sentinel lymph nodes (SLNs) assessed by contrast-enhanced ultrasound (CEUS) in breast cancer patients. World J Surg Oncol 15:127. Received: 17 December 2021 Accepted: 9 March 2022 https://doi.org/10.1186/s12957-017-1195-3 23. 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Journal

European Radiology ExperimentalSpringer Journals

Published: Apr 12, 2022

Keywords: Contrast media; Injections (intradermal); Lymphatic vessels; Sonazoid; Ultrasonography

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