Postoperative control of functional muscle flaps for facial palsy reconstruction: Ultrasound guided tissue monitoring using contrast enhanced ultrasound (CEUS) and ultrasound elastography

Abstract

BACKGROUND AND OBJECTIVES:

Facial paralysis causes excruciating impairments including facial asymmetry, limited eye closure, oral incontinence and social dysfunction. Modern plastic surgical reconstructions render favorable results with well-perfused dynamic muscle flaps. Post-operative tissue monitoring is a critical determinant for success. Contrast-enhanced Ultrasound (CEUS) and elastography have proven superior properties to evaluate tissue perfusion in various organs. We evaluated their role for functional muscle flaps positioned at the sub-skin level in facial palsy patients.

METHODS:

From 2016–2017 five patients received muscle flap reconstructions. Flaps included four free transplants and one pedicled transfer. Postoperatively tissue vitality and blood flow were assessed with CEUS. One experienced examiner using linear probes (6–9, 6–15 MHz) and bolus injections of Sulphur-hexafluoride microbubbles evaluated tissue perfusion. Using the time intensity curve- (TIC)-analysis measurements were recorded for TTP (time to peak) and AUC (Area under curve). Tissue elasticity was assessed with ultrasound elastography.

RESULTS:

All flaps were successful and showed no major complications. TTP-values in flap tissue showed slightly decreased values of 35.12±33.99 s and 25.04±10.86 s compared to surrounding tissue with 19.88±6.94 s. AUC-analysis however revealed higher values of 292.25±169.52 RU and 274.51±115.88 RU than surrounding tissue with 150.90±40.21 RU. Elastography demonstrated predominantly elastic flap tissue whereas surrounding tissue confirmed a slightly harder tissue quality. CEUS in combination with elastography verified tissue vitality and blood flow in a safe and reproducible manner.

CONCLUSIONS:

Post-operative perfusion monitoring in muscle flaps positioned at a sub-skin level may be performed superiorly by CEUS and elastography in a quick, reproducible and minimally-invasive fashion.

Keywords

Facial paralysis functional muscle flaps contrast-enhanced ultrasound (CEUS)elastography tissue perfusion monitoring blood flow

ABBREVIATIONS

CEUS

contrast-enhanced ultrasound

TTP

time to peak (Vmax)

AUC

area under curve (Volume)

TIC-analysis

time intensity curve-analysis

CCDS

color-coded-duplex-sonography

1 Introduction

Facial expression is determined through a complex dynamic neuromotor and psychomotor interplay which channels emotions into involuntary and voluntary mimic movements [1]. The facial nerve represents a key protagonist linking the brain with at least 21 mimic muscles. Dysfunction of the nerve may be congenital or acquired. Long-standing facial paralysis may cause excruciating impairments for affected patients including facial asymmetry, limited eye closure, oral incontinence and social dysfunction [2]. Modern plastic surgical reconstructions offer favorable results with well-perfused dynamic muscle flaps aiming at reanimating the face [3 –7]. In this context post-operative flap tissue monitoring for perfusion and vitality is a critical determinant for clinical success to achieve functional and aesthetic restoration.

Perfusion monitoring via clinical characteristics as capillary refill, color, temperature and turgor is precluded by a lack of an intrinsic surface skin island as free functional muscle transplants as well as pedicled muscle flaps are usually protected by facial skin.

Current ultrasound guidelines for contrast enhanced sonography and elastography underline excellent properties for a high-resolution visualization of flap tissue regarding morphology, elasticity, vascularization and perfusion [8 –10]. Contrast-enhanced Ultrasound (CEUS) has proven superior properties to evaluate tissue perfusion and blood volume in various organs featuring a minimally invasive approach [11 –15].

Relating to microvascular tissue transplants the significance of a time intensity curve-analysis (TIC-analysis) could already be demonstrated [16 –18]. Bolus-contrast enhancement could be optimized for tissue evaluation starting in an early arterial phase (15–25 s) to a venous tissue vascularisation (−1 min). The technique of Contrast-harmonic-Imaging (CHI) is used with a low mechanical Index (low MI <0.2) intravenously injecting Sulfurhexaflorid-micro bubbles, a second-generation contrast agent (SonoVue®, Bracco, Italy) [19]. Initially deployed for liver assessment this method has been meanwhile successfully established for a multitude of other tissue evaluations including vascular [20, 21].

The relevance of high-resolution ultrasound diagnostics has further been described for the peripheral nervous system, e.g. detailed visualizations of neural structures post-operatively [22]. However, complex ultrasound technology applying high frequency probes paired with advanced examiner skills are mandatory requirements for this method.

Post-operative monitoring of flap reconstructions featuring a positioning of functional muscle grafts profound to the skin surface as in facial palsy surgery requires a combination of both methods. Visualization should include high-resolution tissue morphology, tissue elasticity and dynamic contrast enhanced tissue perfusion monitoring.

In this pilot study of a small series of patients with chronic facial paralysis we evaluated the role of CEUS in combination with elastography for functional muscle flaps positioned at the sub-skin level. We hypothesized that a superior level of tissue perfusion monitoring in functional muscle transplants could be achieved with combining advantages of both ultrasound methods.

2 Methods

2.1 Surgical reconstruction of long-standing facial paralysis

All patients included into the study were operated in the Department of Plastic, Hand and Reconstructive Surgery at the University Hospital Regensburg/Germany by an experienced surgeon. From 2016–2017 five patients with long standing facial paralysis received functional muscle flap reconstructions. Flaps included four free muscle transplants and one pedicled muscle transfer. Surface monitoring through clinical perfusion tests was precluded by a lack of skin components and sub-skin flap positioning in the face in all cases (Fig. 1).

Fig.1

Transplantation of a free functional Latissimus-dorsi-(LD)-muscle for facial reanimation in a 33 year old male patient with chronic facial palsy (no.2). Situation of LD-muscle in situ immediately after microsurgical anastomoses of the neurovascular pedicle to the facial artery and vein and nerve coaptation to the cross-face-nerve-graft (left picture). End of plastic-surgical reconstruction after skin flap coverage of the muscle flap (right picture).

2.2 Post-operative ultra-sound guided tissue monitoring

Therefore, tissue vitality and blood flow were assessed with ultrasound diagnostics postoperatively employing CEUS in combination with compound as well as shear wave elastography in all patients. The study was approved by the Institutional Review Board Committee and was designed in accordance with the Declaration of Helsinki. All patients approved of the operation as well as our subsequent tissue monitoring study with contrast by informed consent.

The ultrasound sequence comprised different methodological steps. A single experienced examiner using linear multifrequency probes 6–9 MHz and 6–15 MHz (LOGIQ E9/GE) performed the examination. First tissue morphology of skin, subcutaneous and flap muscle tissue as well as surrounding structures were assessed. Using the color-coded-Doppler sonopgraphy (CCDS) mode the neurovascular pedicle and arterial and venous vessels intrinsic to the muscle flap tissue were evaluated for patent blood flow. Power-Doppler-Mode was used if vascular diameter was of limited dimension. These steps were followed by compound as well as shear wave elastography evaluating for areas of homogeneity and inhomogeneity in the tissue. Scanning comprised any local or generalized indurations in contrast to soft homogenous tissue characteristics. Whereas values of <1,5 m/s were defined as elastic tissue (not hardened), values of >3 m/s were defined as fibrotic tissue with severe hardening. For further differentiation findings were classified into a score system ranging from 5 points (evenly homogenous tissue) to 1 point (very indurated tissue).

Subsequently, after application of bolus injections of 1–2.4 ml Sulphur-hexafluoride microbubbles (SonoVue^®/Bracco, Italy) as a contrast agent to amplify echogenity microcirculation of the flap center, flap edges and surrounding tissue focusing with Regions of Interest (ROIs) was evaluated. Flash-kinetics using a MI up 1 for less than 1 sec, followed after 1 Min for evaluation the replenishment and also the arterial enhancement also in others regions of the tissue transplant. CEUS was performed in a Low Mi-technique (MI < 0–2). Tissue perfusion was scrutinized with cine loops from 15 sec up to 3 Min and digitally recorded. Focusing on the center flap tissue, continuously scanning for one minute after injection arterial vascularization could be noted after 10–15 s (6–9 Mhz probe). Thereafter performing the sweep technique the whole area of the operated facial half was examined for necrosis, hematoma or fluid collections. Neck vessels were delineated bilaterally to rule out thrombi and stenoses.

In the second part of the study digital pictures were anonymously evaluated with TIC (time-intensity-curve) analysis. One region of interest (ROI) was placed in flap center, one at the flap border and one further ROI in surrounding tissue to the flap respectively (Figs. 2 and 3). Measurements were recorded for TTP (time to peak) and AUC (Area under curve).

Fig.2

TIC-Analysis of patient no. 3. ROIs round shaped: red = flap center, yellow = flap peripheral, green = surrounding soft tissue. Dynamic contrast-enhanced ultrasound examination (intravenous bolus injection of 2.4 ml SonoVue^®): Visualization of perfusion over 1 min of time with TIC-analysis. Immediate adequate contrast accumulation starting after 10 seconds. Well-perfused tissue quality with AUC-values of 200–496 RU. Examination with multifrequency linear probe (6–9 MHz).

Fig.3

TIC-Analysis of patient no. 4. ROIs ellipsoid shaped: yellow = flap center, green = flap peripheral, red = surrounding soft tissue. Increase around 20 seconds. Perfusion values over 100 RU. Regions of Interest were selected larger (1×3 cm) than usual. Intravenous bolus injection of 2,4 ml SonoVue^®. TIC-analysis of 1 minute. Examination with multifrequency linear probe (6–9 MHz).

2.3 Statistics

The data analysis (Mean values and standart deviations) for this paper was generated using Excel^® for Mac (2017), Microsoft.

3 Results

3.1 Clinical results

Two gracilis, one pectoralis minor and one latissimus dorsi muscle flaps were transplanted into the paralyzed facial half for reconstruction (Fig. 1). One patient (no. 3) received a regional muscle transfer of the temporalis muscle. All functional muscle flaps survived. No major surgical complications requiring revision surgery occurred. Additionally, no immediate complications as well as potential secondary sequelae regarding the usage of agent were noted. Two cases showed minor hematomas, little signs of lymph edema, however no necroses of muscle tissue transplants or inflammatory fluid collections were seen.

3.2 Evaluation with CEUS and elastography

Picture quality was sufficient for both interpretation of elastography and CEUS. No arterial stenoses, thrombi, fistula were detected.

TIC-Analysis demonstrated comparable values of flap center tissue and surrounding soft tissue in four patients (pat. No 1–4.) Patient no. 5 showed a delayed tissue perfusion with a TTP of up to 96 sec by prolongated capillary enhancement by inhomgeneous soft tissue with particularly fibrosis. The elastography in this patient showed physiological properties with well-perfused vital tissue. In the majority of the cases (4 cases) measurements over 1 min time resulted in a timely contrast enhancement (Table 1). The AUC-analysis showed comparable/respective/similar values for tissue perfusion as the TTP values suggested. AUC-values of flap center and peripheral flap tissue were higher as surrounding soft tissue (Table 2). The Tic-analysis showed for the flap center a mean value (±standard deviation) of TTP of 35.12 (±33.99)s, peripheral flap of TTP of 25.04 (±10.86)s and surrounding soft tissue of TTP of 19.88(±6,94)s. The AUC showed values of 292.25(±169.52) RU, 25.04 (±10.86) RU and 150.90 (±40.21) RU respectively (Tables 1 and 2).

Table 1
Time to peak evaluation (TTP) of Tic-Analysis in Contrast-Enhanced-Ultrasound (CEUS). perfusion imaging Table featuring Time-to-peak (TTP) values measured in Time Intensity Curve-(Tic)-analysis for ROIs focused in flap center, peripheral flap and surrounding soft tissue employing Contrast-enhanced ultrasound (CEUS). A scoring system was developed to grade different time frames for TTP-values

patient TTP flap center (sec) TTP flap peripheral (sec) TTP soft tissue (sec)

1 18.45 23.65 13.52

2 19.17 43.17 22.57

3 26.11 23.56 19.92

4 16.30 20.78 30.00

5 95.57 14.05 13.36

MW (±SA) 35.12±33.99 25.04±10.86 19.88±6.94

CEUS-score

TIC-Scale/TTP Grading

excellent = 0–10 s

good = 10–20 s

fair = 20–25 s

delayed = 25–30s

insufficient = >30 s

patient	TTP flap center (sec)	TTP flap peripheral (sec)	TTP soft tissue (sec)
1	18.45	23.65	13.52
2	19.17	43.17	22.57
3	26.11	23.56	19.92
4	16.30	20.78	30.00
5	95.57	14.05	13.36
MW (±SA)	35.12±33.99	25.04±10.86	19.88±6.94
CEUS-score
TIC-Scale/TTP Grading
excellent = 0–10 s
good = 10–20 s
fair = 20–25 s
delayed = 25–30s
insufficient = >30 s

Results: TTP flap center: 3× excellent, 1× fair, 1× insufficient; TTP flap peripheral: 1× good, 3× fair, 1× insufficient; TTP soft tissue: 3× good, 1× fair, 1× insufficient.

Table 2

Area-under-the curve-(AUC)-analysis in Contrast-Enhanced-Ultrasound (CEUS) perfusion imaging. Table featuring measured Area-under-curve-(AUC)-values in Time Intensity Curve-(Tic)-analyis for the same ROIs as described in Table 1. A scoring system was developed to grade different RU-ranges according to previous studies [23]

Patient	AUC flap center (RU)	AUC flap peripheral (RU)	AUC soft tissue (RU)
1	303.14	287.59	175.03
2	412.24	221.66	111.08
3	496.09	366.56	203.17
4	162.75	105.36	153.76
5	87.05	391.39	111.44
MW (±SD)	292.25(±169.52)	274.51 (±115.88)	150.90 (±40.21)

AUC-scale/grading: hypoperfused tissue:<100 RU, well-perfused tissue >100 RU, excellent-perfused tissue >200 RU. Results: AUC flap center: 3x excellent-perfused tissue, 1x well-perfused tissue, 1x hypoperfused tissue; AUC flap peripheral: 4x excellent-perfused tissue, 1x well-perfused tissue; AUC soft tissue: 1x excellent-perfused tissue, 4x well-perfused tissue.

The elastographic exam showed soft and well-compressible tissue in all cases (Fig. 4). Indurated or incompressible tissues were absent. One case demonstrated ideal measurements (Table 3). Shear wave elastography demonstrated similar values for tissue elasticity suggesting viable well-vascularized tissue in all five cases (Table 4). Values of compound and shear wave elastography are documented in Tables 3 and 4.

Fig.4

Patient no. 5: B-mode-visualization (left) and compound elastography (right) of a free functional gracilis-muscle-flap positioned in the left facial half and covered by facial skin flap. The muscle flap demonstrates predominantly soft, homogenous tissue coded green and red without circumscribed pathological signs of tissue hardening. Examination with multifrequency linear probe (6–9 MHz). Color spectrum: soft = red, hard = blue.

Table 3

Compound Elastography. Table featuring values of the compound elastography examination referring to a scoring system differentiating five different grades of tissue elasticity over a color spectrum from red (= soft) to blue (= hard). See Fig. 4

Elastography - Scale
5 = homogenous soft
4 = predominantly elastic
3 = partially hardened
2 = predominantly hardened
1 = homogenous hard
patient	flap center	flap peripheral	soft tissue
1	4	3	4
2	4	2	3
3	3	3	3
4	4	3	3
5	4	3	4
MW (SD)	3,80 (±0,45)	2,80 (±0,45)	3,40 (±0,55)

Results: flap center: predominantly elastic quality; flap peripheral: partially hardened quality; surrounding soft tissue: transition between predominately elastic to partially hardened quality.

Table 4

Shear-Wave-Elastography. Table featuring values of the shear wave elastography examination referring to a scoring system differentiating five different grades of tissue elasticity over a color spectrum from red (= soft) to blue (= hard)

patient	Flap center	Flap peripheral	Soft tissue
1	4	3	4
2	4	2	3
3	3	3	3
4	4	3	3
5	4	4	3
MW (SD)	3,80 (±0,45)	3,00 (±0,71)	3,20 (±0,45)

Results: flap center: predominantly elastic quality; flap peripheral: anteilig partially hardened quality; surrounding soft tissue: partially hardened quality.

4 Discussion

4.1 Combining B-Mode and CCDS with CEUS and elastography

Acquiring an orientation of tissue homogeneity the results of this retrospective study suggest that compound elastography is helpful assessing vitality through hardened and soft tissue areas within microsurgical transplanted muscle flaps and regional muscle transfer employing a visualization of elastic and non-elastic tissue properties. B-mode ultrasound is indispensable for detection of localized hematoma formations and discrimination of regularities/irregularities of muscle fibres and possibly associated nerves. Using high-resolution probes Doppler and Color-Duplex-Ultrasound maintain their importance to visualize extracranial vessels, vascular anastomoses with venous and arterial flow. However, regarding subcutanous functional muscle flaps the required characteristics can be evaluated with CCDS only in a limited fashion.

For this indication CEUS employing dynamic properties offers a much more detailed evaluation of tissue vitality and perfusion [15]. Artery and vein within the vascular pedicle of the flap as well as hyper- and hypoperfused tissue areas within the muscle transplant can be assessed regarding a timely, untimely or too early dynamic inflow. Using the qualitative TIC-analysis hyperemia (too early inflow) or partial hypoperfusion can be easily detected [16].

4.2 Confirmation of previous findings in TIC-analysis

In accordance with previous data described by our science group and referring to international guidelines of exams of 1 min time the reference values of well-perfused tissue are between 100–300 RU. Well-perfused flap tissue shows >100 RU, ideal perfused tissue >200 RU in AUC-Exams [16 , 24]. This fact could be further supported by this study.

Delayed perfusion below a cut-off value of 50 RU should be interpreted as critical. Values above 500 RU may signify a present hyperemia, reactive inflammatory tissue response of origin or expression of AV-shunting.

In the clinical functional muscle flap series of the present pilot study no cases of complete or extensive local necroses were seen. Good results in elastography were correlated with good clinical results. Remarkably, TTP- as well as AUC-values of center flap and peripheral flap tissue were higher as in surrounding soft tissue ROIs. This circumstance could demonstrate a slightly higher perfusion in muscle flap tissue post-operatively.

Small localized hematoma formations may cause minor reduction of tissue graft perfusion, however do not reach a critical extend. Inflammatory fluid collections were not detected either. In accordance with earlier results of our group it could be demonstrated that besides an arterial inflow a continuous venous outflow is decisive for a successful outcome [23]. If venous stasis is present, tissue edema and in extreme cases proportionate thrombi formation are seen and may subsequently cause tissue hypoperfusion. In patient No. 4 the surrounding soft tissue showed a slow increase of inflow which was interpreted with limited soft tissue perfusion associated with heavy smoking. Thus the facial skin flap covering the transplanted functional gracilis muscle graft demonstrated limited perfusion properties.

Requiring this complex judgement of tissue vitality a combination of CEUS and elastography are diagnostically constructive, as necroses are hardened, but edema soft and correspondingly results in different color coding. Furthermore shear wave elastography was used to measure functional muscle flaps for the first time. Combination of hemodynamics with CCDS, quantification of tissue elasticity with shear wave, compressibility with compound, elastography with CEUS in liaison with TIC-analysis are representing an intricate diagnostic sequence, however distinctly offering enhanced diagnostic opportunities of a precise and detailed tissue assessment in follow-up of complex tissue reconstructions. This opens broad fields for prospective, multicenter evaluations of tissue reconstructions. So far our study sample represent a small pilot study, therefore further prospectively designed, multicenter studies have to be conducted to further elucidate and support our data.

5 Conclusion

Adversities suffered from persistent malfunction of the facial nerve may successfully be eased with plastic surgical transfer or transplantation of muscle flaps. Prerequisite for favorable functional results are tissue vitality and adequate perfusion.

Modern high resolution ultrasound techniques in combination with elastography and CEUS enable an excellent real time imaging modality for monitoring a special kind of tissue and nerval transplants.

Disclosure and funding sources

There were no sources of support in the form of grants, equipment, drugs or of other nature. No conflicts of interest.

Footnotes

Acknowledgments

The authors thank Simon Engelmann and Robert Bauer of the University of Regensburg for their contribution to the scientific background study of the facial paralysis program at the University of Regensburg.

References

Garcia

R.M.

, Hadlock

T.A.

, Klebuc

M.J.

, Simpson

R.L.

, Zenn

M.R.

and Marcus

J.R.

, Contemporary solutions for the treatment of facial nerve paralysis, Plast Reconstr Surg135(6) (2015), 1025e–1046e.

Kosins

A.M.

, Hurvitz

K.A.

, Evans

G.R.

and Wirth

G.A.

, Facial paralysis for the plastic surgeon, Can J Plast Surg15(2) (2007), 77–82.

Woollard

A.C.

, Harrison

D.H.

and Grobbelaar

A.O.

, An approach to bilateral facial paralysis, J Plast Reconstr Aesthet Surg63(9) (2010), 1557–1560.

Terzis

J.K.

and Olivares

F.S.

, Long-term outcomes of free-muscle transfer for smile restoration in adults, Plast Reconstr Surg123(3) (2009), 877–888.

Rozen

S.M.

, Facial Reanimation: Basic Surgical Tools and Creation of an Effective Toolbox for Treating Patients with Facial Paralysis. Part A: Functional Muscle Transfers in the Long-Term Facial Palsy Patient, Plast Reconstr Surg139(2) (2017), 469–471.

Gousheh

and Arasteh

, Treatment of facial paralysis: Dynamic reanimation of spontaneous facial expression-apropos of 655 patients, Plast Reconstr Surg128(6) (2011), 693e–703e.

Chuang

D.C.

, Lu

J.C.

and Anesti

, One-stage procedure using spinal accessory nerve (XI)-innervated free muscle for facial paralysis reconstruction, Plast Reconstr Surg132(1) (2013), 117e–129e.

Dietrich

C.F.

, Bamber

, Berzigotti

, Bota

, Cantisani

, Castera

, et al., EFSUMB Guidelines and Recommendations on the Clinical Use of Liver Ultrasound Elastography, Update 2017 (Long Version), Ultraschall Med2017.

Nylund

, Maconi

, Hollerweger

, Ripolles

, Pallotta

, Higginson

, et al., EFSUMB Recommendations and Guidelines for Gastrointestinal Ultrasound, Ultraschall Med38(3) (2017), e1–e15.

10.

Sporea

, The new EFSUMB Guidelines on Liver Elastography 2017: Why and for whom?Med Ultrason19(1) (2017), 5–6.

11.

Yue

W.W.

, Wang

, Xu

H.X.

, Sun

L.P.

, Guo

L.H.

, Bo

X.W.

, et al., Parametric imaging with contrast-enhanced ultrasound for differentiating hepatocellular carcinoma from metastatic liver cancer, Clin Hemorheol Microcirc64(2) (2016), 177–188.

12.

Schellhaas

, Waldner

M.J.

, RS

G.O.

, Vitali

, Kielisch

, Pfeifer

, et al., Diagnostic accuracy and interobserver variability of Dynamic Vascular Pattern (DVP) in primary liver malignancies - A simple semiquantitative tool for the analysis of contrast enhancement patterns, Clin Hemorheol Microcirc2017. doi: 10.3233/CH-16238

13.

Jung

E.M.

, Platz

, da Silva

Batista

, Jung

, Farkas

, Stroszczynski

, Rennert

, et al., Is Strain Elastography (IO-SE) Sufficient for Characterization of Liver Lesions before Surgical Resection–Or Is Contrast Enhanced Ultrasound (CEUS) Necessary?PLoS One10(6) (2015), e0123737.

14.

Huf

, Platz da Silva Batista

, Wiesinger

, Hornung

, Scherer

M.N.

, Lang

, et al., Analysis of Liver Tumors Using Preoperative and Intraoperative Contrast-Enhanced Ultrasound (CEUS/IOCEUS) by Radiologists in Comparison to Magnetic Resonance Imaging and Histopathology, Rofo189(5) (2017), 431–440.

15.

Geis

, Prantl

, Schoeneich

, Lamby

, Klein

, Dolderer

, et al., Contrast enhanced ultrasound (CEUS) - an unique monitoring technique to assess microvascularization after buried flap transplantation, Clin Hemorheol Microcirc62(3) (2016), 205–214.

16.

Jung

E.M.

, Clevert

D.A.

, Schreyer

A.G.

and Schmitt

, Rennert

, Kubale

, et al., Evaluation of quantitative contrast harmonic imaging to assess malignancy of liver tumors: A prospective controlled two-center study, World J Gastroenterol13(47) (2007), 6356–6364.

17.

Geis

, Schreml

, Lamby

, Obed

, Jung

E.M.

, Nerlich

, et al., Postoperative assessment of free skin flap viability by transcutaneous pO2 measurement using dynamic phosphorescence imaging, Clin Hemorheol Microcirc43(1-2) (2009), 11–18.

18.

Geis

, Prantl

, Mueller

, Gosau

, Lamby

and Jung

E.M.

, Quantitative assessment of bone microvascularization after osteocutaneous flap transplantation using contrast-enhanced ultrasound (CEUS), Ultraschall Med34(3) (2013), 272–279.

19.

Greis

, [Summary of technical principles of contrast sonography and future perspectives], Radiologe51(6) (2011), 456–461.

20.

Wendl

C.M.

, Eiglsperger

, Schuierer

and Jung

E.M.

, Evaluating post-interventional occlusion grades of cerebral aneurysms with transcranial contrast-enhanced ultrasound (CEUS) using a matrix probe, Ultraschall Med36(2) (2015), 168–173.

21.

Rubenthaler

, Paprottka

K.J.

, Hameister

, Hoffmann

, Joiko

, Reiser

, et al., Diagnostic accuracy of contrast-enhanced ultrasound (CEUS) in monitoring vascular complications in patients after liver transplantation - diagnostic performance compared with histopathological results, Clin Hemorheol Microcirc (2017). doi: 10.3233/CH-179105

22.

Girtler

M.T.

, Krasinski

, Dejaco

, Kitzler

H.H.

, Cui

L.G.

, Sherebrin

, et al., Feasibility of 3D ultrasound to evaluate upper extremity nerves, Ultraschall Med34(4) (2013), 382–387.

23.

Geis

, Prantl

, Dolderer

, Lamby

, Mueller

and Jung

E.M.

, Postoperative monitoring of local and free flaps with contrast-enhanced ultrasound (CEUS)–analysis of 112 patients, Ultraschall Med34(6) (2013), 550–558.

24.

Geis

, Klein

, Prantl

, Dolderer

, Lamby

and Jung

E.M.

, Quantitative Assessment of Free Flap Viability with CEUS Using an Integrated Perfusion Software, Handchir Mikrochir Plast Chir47(6) (2015), 389–395.