Non-invasive Molecular Imaging to Determine Hemoglobin and Collagen Levels in Muscles Before and After Exercise, and Over Time
Phase: NA
Conditions: Muscular Diseases
Sponsor: University of Erlangen-Nürnberg Medical School

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Non-invasive molecular imaging to determine the hemoglobin and collagen 
content in muscles before and after physical exercise and over time 
(MSOT_muscles) 
 
 
Unique Protocol ID: 169_19B 
ClinicalTrials.gov Identifier: NCT03979157 
Version date: 23.04.2019 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Sponsor: 
Department of Pediatrics and Adolescent Medicine 
University Hospital Erlangen 
Friedrich-Alexander-University (FAU) Erlangen-Nürnberg 
Pediatric Experimental and Translational Imaging Laboratory (PETI-Lab) 
Loschgestraße 15 
91054 Erlangen, Germany  
Tel: +49 9131 85 33118  
 
 

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Study protocol 
 
 
 
MSOT_muscles 
 
Non-invasive molecular imaging to determine the hemoglobin and collagen content in 
muscles before and after physical exercise and over time. 
 

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1. Table of contents 
 
1. 
Table of contents ............................................................................................................ 3 
2. 
Study title, version number, version date ........................................................................ 4 
3. 
Summary of the project .................................................................................................. 5 
4. 
Responsabilities ............................................................................................................. 7 
5. 
Scientific background ..................................................................................................... 8 
6. 
Study objectives ............................................................................................................13 
7. 
Target parameters .........................................................................................................16 
8. 
Study design .................................................................................................................17 
9. 
Study population............................................................................................................19 
10. 
Study course ..............................................................................................................20 
11. 
Risk-benefit analysis ..................................................................................................23 
12. 
Biometrics ..................................................................................................................26 
13. 
Data management und and data protection ...............................................................27 
14. 
Handling of biomaterials ............................................................................................28 
15. 
Insurance ...................................................................................................................28 
16. 
Signatures .................................................................................................................29 
 
 
 

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2. Study title, version number, version date 
Study title  
Non-invasive molecular imaging to determine the hemoglobin and collagen content in 
muscles before and after physical exercise and over time. 
 
Version number 
Version 1.2 
 
Version date 
23.04.2019 
 
 

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3. Summary of the project 
Since 2017, a Multispectral Optoacoustic Tomograph (MSOT) with an extended emission 
spectrum is available to the University Hospital Erlangen through a DFG large-scale equipment 
funding. For MSOT imaging, similar to conventional sonography a transducer is placed on the 
skin, and instead of sound, energy is applied to the tissue by means of pulsed laser light. This 
leads to a constant change of minimal expansion and contraction of individual tissue 
components or molecules. The resulting sound waves can then be detected by the same 
device. Previous studies have already shown that the quantitative determination of 
haemoglobin can provide information on blood flow and inflammatory activity in chronic 
inflammatory bowel diseases. In the extended spectrum other markers such as collagens and 
lipids can be detected in addition to haemoglobin and its oxygenation status. We were able to 
show with our experimental preliminary work and a large animal pig model that an in vivo 
quantification of collagen is possible. In the first application of MSOT in children with Duchenne 
muscular dystrophy, the measured collagen content correlated with the clinical function tests. 
In summary, we could show that the molecular muscle structure quantified by MSOT allows 
the establishment of new, age-independent, non-invasive molecular biomarkers for disease 
assessment and therapy monitoring in patients with DMD. 
 
Neuromuscular diseases often present as early as in neonatal age and manifest themselves 
in muscular hypotonia and weakness. Associated diseases are caused by numerous 
pathologies in the central nervous system (brain and spinal cord), the peripheral nervous 
system or skeletal muscle. X-linked progressive muscular dystrophy of the Duchenne type 
(DMD) is one of the most common progressive muscular diseases of childhood with an 
incidence of 1:3500 male newborns and is primarily associated with a reduced life expectancy. 
Around the age of 4-5 years, motor problems manifest themselves in everyday life with typical 
signs of proximal muscle weakness and laboratory chemical increase of the muscle enzyme 
(creatinine kinase, CK). Within a few years, relevant muscle and tendon shortenings develop, 
leading to joint malpositions, instabilities, and consecutively to scoliosis and loss of walking 
around the age of 10. Supportive therapy measures cannot curatively influence complications 
and progression of the disease. Pathogenetically, there is a deficiency of dystrophin, a 
structural protein of the sarcolemma, which is caused by mutations (mostly deletions) of the 
dystrophin gene (Xp21.3-p21.2). The consequence of dystrophin deficiency is necrosis of the 
muscle cells which are replaced by connective tissue and fat. The 6-minute walk test (6-MWT) 
is usually used to quantitatively assess the disease progression as well as the effects of 
therapy. In this test, the patient need to walk as much distance as possible within 6 minutes. 
However, this test is dependent on cooperation and is usually not successful in the very young 
patients. The effects of new gene therapeutic therapies (e.g. PTC 124) are currently still being 

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assessed with the help of this clinical function test. Sensitive, non-invasive methods for 
measuring muscle degeneration at an early stage and muscle function during the course of 
the disease are therefore of great clinical and scientific importance. 
The MSOT data collected so far seem promising to enable therapy monitoring in DMD patients. 
Preliminary work by other groups has already demonstrated a very good precision and 
reproducibility of MSOT. However, it has not yet been investigated to what extent 1) previous 
physical activity, 2) different measurement positions of the muscle, 3) different examination 
dates and 4) intra- and interrater variability influence the measurement results.  Especially 
molecular changes immediately after the 6-MWT are relevant for studies in muscle patients 
with DMD.  
In this first pilot study, the aim is to investigate in healthy adults whether increased exercise 
alters haemoglobin values, the collagen content remains constant and repeated 
measurements at different muscle positions by different investigators provide consistent 
measurement results over time. This information is essential for further studies of children with 
neuromuscular disorders to properly evaluate a possible response to therapy. 
 
 
 
 
 
 

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4. Responsabilities 
 
Study lead 
Dr. Adrian Regensburger 
Pediatric Experimental and Translational Imaging Laboratory (PETI_Lab) 
Department of Pediatrics and Adolescent Medicine  
University Hospital Erlangen 
Friedrich-Alexander-University (FAU) Erlangen-Nuremberg 
Loschgestr. 15, 91054 Erlangen, Germany 
Tel. 09131 8533118 
Mail: adrian.regensburger@uk-erlangen.de 
Dr. Ferdinand Knieling 
Pediatric Experimental and Translational Imaging Laboratory (PETI_Lab) 
Department of Pediatrics and Adolescent Medicine  
University Hospital Erlangen 
Friedrich-Alexander-University (FAU) Erlangen-Nuremberg 
Loschgestr. 15, 91054 Erlangen, Germany 
Tel. 09131 8533118 
Mail: ferdinand.knieling@uk-erlangen.de  
Prof. Dr. Regina Trollmann 
Head of the Department of Neuropaediatrics 
Department of Pediatrics and Adolescent Medicine  
University Hospital Erlangen 
Friedrich-Alexander-University (FAU) Erlangen-Nuremberg 
Loschgestr. 15, 91054 Erlangen, Germany 
Tel.: 09131 85 33753 
Mail: regina.trollmann@uk-erlangen.de 
Other facilities possibly involved (e.g. laboratory, imaging, etc.)  
Prof. Dr. Maximilian J. Waldner (technical realization)  
Department of Medicine 1   
University Hospital Erlangen  
Friedrich-Alexander-University (FAU) Erlangen-Nuremberg  
Ulmenweg 18, 91054 Erlangen, Germany   
Tel.: 09131 8545025  
Mail: maximilian.waldner@uk-erlangen.de 
 
Sponsoring   
Children’s Hospital Erlangen, Else-Kröner-Fresenius Foundation (Else-Kröner-Fresenius 
Memorial Scholarship Ferdinand Knieling)  

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5.  Scientific background 
Since 2017, the University Hospital Erlangen (Medical Clinic 1, Pediatric and Adolescent 
Clinic) has a Multispectral Optoacoustic Tomograph (MSOT) funded by the DFG. This allows 
non-invasive, quantitative imaging of the composition of target tissues in the non-sedated child, 
comparable to sonography. This is of particular importance in this patient group. 
 
In MSOT, similar to conventional 
sonography, 
a 
transducer 
is 
placed on the skin and instead of 
sound, energy is supplied to the 
tissue by means of pulsed laser 
light. This leads to a constant 
change of minimal expansions 
and contractions (thermoelastic 
expansion) of individual tissue 
components or molecules. The 
resulting sound waves can then be 
detected by the same examination 
unit. Previous studies have shown 
that the quantitative determination 
of hemoglobin can be used to 
obtain information on blood flow 
and inflammatory activity in the 
intestine 
from 
patients 
with 
Crohn's 
disease 
(Waldner, 
Knieling et al. 2016, Knieling, 
Neufert et al. 2017) (Waldner 
2016, Knieling 2017). In the newly 
configured device (Acuity Echo, 
iThera 
Medical 
GmbH, 
Munich, 
prototype) an extended spectrum of laser light can be used, which ultimately not only enables 
the detection of haemoglobin and its oxygenation stages, but also the detection of further 
markers such as collagen and lipid. This principle has already been successfully used in a 
follow-up study on pediatric patients with Duchenne muscular dystrophy (Regensburger, 
Fonteyne et al., submitted for publication, ESMI Young Investigator Award 2019). 
 
 

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We were able to show by our experimental preliminary work and a large animal pig model that 
an in vivo quantification of collagen is possible. In the first application of MSOT in children with 
Duchenne muscular dystrophy, the measured collagen content correlated with the clinical 
function tests. In summary, we could show that the molecular muscle structure quantified by 
MSOT allows the establishment of new, age-independent, non-invasive molecular biomarkers 
for disease assessment and therapy monitoring in patients with DMD. 
 
Neuromuscular diseases often present as 
early as in neonatal age and manifest 
themselves in muscular hypotonia and 
weakness. Associated diseases are caused 
by numerous pathologies in the central 
nervous system (brain and spinal cord), the 
peripheral nervous system or skeletal 
muscle. X-linked progressive muscular 
dystrophy of Duchenne type (DMD) is one 
of the most common progressive muscular 
diseases of childhood with an incidence of 
1:3500 male newborns and is primarily 
associated with a reduced life expectancy 
(Mendell and Lloyd-Puryear 2013). From 
the age of 4-5 years, motor problems 
manifest in everyday life with typical signs of 
proximal muscle weakness and laboratory chemical increase of the muscle enzyme (creatinine 
kinase, CK). Within a few years, relevant muscle and tendon shortenings develop, leading to 
joint malpositions, instabilities, and scoliosis and loss of walking around the age of 10. 
Supportive therapy measures cannot curatively influence complications and progress of the 
disease. Pathogenetically, there is a deficiency of dystrophin, a structural protein of the 
sarcolemma, which is caused by mutations (mostly deletions) of the dystrophin gene (Xp21.3-
p21.2). The consequence of dystrophin deficiency is necrosis of the muscle cells which are 
replaced by connective tissue and fat (Hoffman, Brown et al. 1987, Klingler, Jurkat-Rott et al. 
2012, Mercuri and Muntoni 2013). The 6-minute walk test (6-MWT) (McDonald, Henricson et 
al. 2010, McDonald, Henricson et al. 2010, Bushby, Finkel et al. 2014, Mendell, Goemans et 
al. 2016, McDonald, Campbell et al. 2017) is usually used for the quantitative assessment of 
the disease progression as well as therapy effects. Here the patient needs to walk the longest 
distance possible within 6 minutes.  
 
Figure 1 – Feasibility of 3D MSOT image. 

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However, this test is dependent on cooperation and is usually not successful in very young 
patients. In addition, daily fluctuations in the patients' function and strength as well as learning 
effects can influence the results. A problematic and limiting factor for the use of the 6MWT as 
a reliable marker is that progressive muscle degeneration is only detected functionally at a late 
stage. MRI studies to characterize the degenerative changes in early-stage skeletal muscles 
show promising results in order to find quantitative measures to determine edematous changes 
and fatty degeneration (Arpan, Willcocks et al. 2014, Bonati, Hafner et al. 2015, Glemser, 
Jaeger et al. 2017, Barnard, Willcocks et al. 2018). For clinical use, the required sedation of 
the patients may be limiting for the feasibility of MRI diagnostics. The highly sensitive 
assessment of therapeutic effects is expected to become increasingly important in the future. 
In the meantime, the detection of a nonsense point mutation leading to a premature stop codon 
and thus to the premature termination of protein synthesis has therapeutic consequences. For 
walkable boys with DMD with a proven nonsense mutation, a gene therapy approach with PTC 
124 (Translarna®, Ataluren, PTC Therapeutics) has been available since 2016 which leads 
via ribosomal read-through to the formation of functionally active full-length dystrophin and to 
a significant improvement of muscle function (Bushby, Finkel et al. 2014, McDonald, Campbell 
et al. 2017). Methods for assessing the efficacy are controversially discussed and are not 
standardized established in clinical practice. Sensitive, non-invasive methods for recording 
muscle degeneration at an early stage and muscle function during its course are therefore of 
great clinical and scientific importance. 
 
The MSOT data collected so far seem promising to enable therapy monitoring in DMD patients. 
Preliminary work by other groups has already demonstrated a very good precision (Joseph, 
Tomaszewski et al. 2017) and reproducibility (Helfen, Masthoff et al. 2019) of MSOT 
measurements (with a technically slightly different prototype). However, no investigations have 
been carried out so far: 
- to what extent physical activity influences the measurement results 
- the extent to which the MSOT values vary within a muscle (different muscle positions) 
- whether the measured values change over time, and 
- whether the intra- and interrater differences also apply to this MSOT prototype   
 
In this first pilot study, the aim is to investigate in healthy adults whether increased exercise 
changes the haemoglobin values, the collagen content remains constant and repeated 
measurements at different positions and by different examiners over time provide consistent 
measurement results. This information is essential for planning further studies in children with 
neuromuscular disorders, and to be able to evaluate a possible response to therapy. 
 

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Literature 
Arpan, I., R. J. Willcocks, S. C. Forbes, R. S. Finkel, D. J. Lott, W. D. Rooney, W. T. Triplett, C. R. 
Senesac, M. J. Daniels, B. J. Byrne, E. L. Finanger, B. S. Russman, D. J. Wang, G. I. Tennekoon, G. A. 
Walter, H. L. Sweeney and K. Vandenborne (2014). "Examination of effects of corticosteroids on 
skeletal muscles of boys with DMD using MRI and MRS." Neurology 83(11): 974-980. 
 
Barnard, A. M., R. J. Willcocks, E. L. Finanger, M. J. Daniels, W. T. Triplett, W. D. Rooney, D. J. Lott, S. 
C. Forbes, D. J. Wang, C. R. Senesac, A. T. Harrington, R. S. Finkel, B. S. Russman, B. J. Byrne, G. I. 
Tennekoon, G. A. Walter, H. L. Sweeney and K. Vandenborne (2018). "Skeletal muscle magnetic 
resonance biomarkers correlate with function and sentinel events in Duchenne muscular dystrophy." 
PLoS One 13(3): e0194283. 
 
Bonati, U., P. Hafner, S. Schadelin, M. Schmid, A. Naduvilekoot Devasia, J. Schroeder, S. Zuesli, U. 
Pohlman, C. Neuhaus, A. Klein, M. Sinnreich, T. Haas, M. Gloor, O. Bieri, A. Fischmann and D. Fischer 
(2015). "Quantitative muscle MRI: A powerful surrogate outcome measure in Duchenne muscular 
dystrophy." Neuromuscul Disord 25(9): 679-685. 
 
Bushby, K., R. Finkel, B. Wong, R. Barohn, C. Campbell, G. P. Comi, A. M. Connolly, J. W. Day, K. M. 
Flanigan, N. Goemans, K. J. Jones, E. Mercuri, R. Quinlivan, J. B. Renfroe, B. Russman, M. M. Ryan, M. 
Tulinius, T. Voit, S. A. Moore, H. Lee Sweeney, R. T. Abresch, K. L. Coleman, M. Eagle, J. Florence, E. 
Gappmaier, A. M. Glanzman, E. Henricson, J. Barth, G. L. Elfring, A. Reha, R. J. Spiegel, W. O'Donnell 
M, S. W. Peltz, C. M. McDonald and G. Ptc124-Gd-007-Dmd Study (2014). "Ataluren treatment of 
patients with nonsense mutation dystrophinopathy." Muscle Nerve 50(4): 477-487. 
 
Glemser, P. A., H. Jaeger, A. M. Nagel, A. E. Ziegler, D. Simons, H. P. Schlemmer, F. Lehmann-Horn, K. 
Jurkat-Rott and M. A. Weber (2017). "(23)Na MRI and myometry to compare eplerenone vs. 
glucocorticoid treatment in Duchenne dystrophy." Acta Myol 36(1): 2-13. 
 
Helfen, A., M. Masthoff, J. Claussen, M. Gerwing, W. Heindel, V. Ntziachristos, M. Eisenblatter, M. 
Kohler and M. Wildgruber (2019). "Multispectral Optoacoustic Tomography: Intra- and Interobserver 
Variability Using a Clinical Hybrid Approach." J Clin Med 8(1). 
 
Hoffman, E. P., R. H. Brown, Jr. and L. M. Kunkel (1987). "Dystrophin: the protein product of the 
Duchenne muscular dystrophy locus." Cell 51(6): 919-928. 
 
Joseph, J., M. R. Tomaszewski, I. Quiros-Gonzalez, J. Weber, J. Brunker and S. E. Bohndiek (2017). 
"Evaluation of Precision in Optoacoustic Tomography for Preclinical Imaging in Living Subjects." J Nucl 
Med 58(5): 807-814. 
 
Klingler, W., K. Jurkat-Rott, F. Lehmann-Horn and R. Schleip (2012). "The role of fibrosis in Duchenne 
muscular dystrophy." Acta Myol 31(3): 184-195. 
 
Knieling, F., C. Neufert, A. Hartmann, J. Claussen, A. Urich, C. Egger, M. Vetter, S. Fischer, L. Pfeifer, A. 
Hagel, C. Kielisch, R. S. Gortz, D. Wildner, M. Engel, J. Rother, W. Uter, J. Siebler, R. Atreya, W. 
Rascher, D. Strobel, M. F. Neurath and M. J. Waldner (2017). "Multispectral Optoacoustic 
Tomography for Assessment of Crohn's Disease Activity." N Engl J Med 376(13): 1292-1294. 
 
McDonald, C. M., C. Campbell, R. E. Torricelli, R. S. Finkel, K. M. Flanigan, N. Goemans, P. Heydemann, 
A. Kaminska, J. Kirschner, F. Muntoni, A. N. Osorio, U. Schara, T. Sejersen, P. B. Shieh, H. L. Sweeney, 
H. Topaloglu, M. Tulinius, J. J. Vilchez, T. Voit, B. Wong, G. Elfring, H. Kroger, X. Luo, J. McIntosh, T. 
Ong, P. Riebling, M. Souza, R. J. Spiegel, S. W. Peltz, E. Mercuri, G. Clinical Evaluator Training and A. D. 
S. Group (2017). "Ataluren in patients with nonsense mutation Duchenne muscular dystrophy (ACT 

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DMD): a multicentre, randomised, double-blind, placebo-controlled, phase 3 trial." Lancet 
390(10101): 1489-1498. 
 
McDonald, C. M., E. K. Henricson, J. J. Han, R. T. Abresch, A. Nicorici, L. Atkinson, G. L. Elfring, A. Reha 
and L. L. Miller (2010). "The 6-minute walk test in Duchenne/Becker muscular dystrophy: longitudinal 
observations." Muscle Nerve 42(6): 966-974. 
 
McDonald, C. M., E. K. Henricson, J. J. Han, R. T. Abresch, A. Nicorici, G. L. Elfring, L. Atkinson, A. Reha, 
S. Hirawat and L. L. Miller (2010). "The 6-minute walk test as a new outcome measure in Duchenne 
muscular dystrophy." Muscle Nerve 41(4): 500-510. 
 
Mendell, J. R., N. Goemans, L. P. Lowes, L. N. Alfano, K. Berry, J. Shao, E. M. Kaye, E. Mercuri, G. 
Eteplirsen Study and D. M. D. I. N. Telethon Foundation (2016). "Longitudinal effect of eteplirsen 
versus historical control on ambulation in Duchenne muscular dystrophy." Ann Neurol 79(2): 257-
271. 
 
Mendell, J. R. and M. Lloyd-Puryear (2013). "Report of MDA muscle disease symposium on newborn 
screening for Duchenne muscular dystrophy." Muscle Nerve 48(1): 21-26. 
 
Mercuri, E. and F. Muntoni (2013). "Muscular dystrophies." Lancet 381(9869): 845-860. 
 
Waldner, M. J., F. Knieling, C. Egger, S. Morscher, J. Claussen, M. Vetter, C. Kielisch, S. Fischer, L. 
Pfeifer, A. Hagel, R. S. Goertz, D. Wildner, R. Atreya, D. Strobel and M. F. Neurath (2016). 
"Multispectral Optoacoustic Tomography in Crohn's Disease: Noninvasive Imaging of Disease 
Activity." Gastroenterology 151(2): 238-240. 
 
 

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6. Study objectives 
The aim of the proposed study is a longitudinal comparison of muscle tissue composition based 
on Multispectral Optoacoustic Tomography (MSOT) of healthy, adult subjects, before and after 
physical exercise. 
 
Primary/secondary objectives and/or hypotheses 
Hypotheses: 
 
The quantitative amount of hemo/myoglobin signal in muscles of healthy volunteers 
determined by MSOT differs before and after the 6-minute walk test. 
 
The quantitative amount of oxygenated/deoxygenated hemo/myoglobin signal in 
muscles of healthy volunteers determined by the MSOT differs before and after the 6-
minute walk test. 
 
The quantitative amount of collagen signal in muscles of healthy volunteers determined 
by MSOT does not differ before and after the 6-minute walk test. 
 
The quantitative amount of hemo/myoglobin signal in muscles of healthy volunteers 
determined by the MSOT does not differ when the follow-up is measured after 2 weeks. 
 
The quantitative proportion of oxygenated/deoxygenated hemo/myoglobin signal in 
muscles of healthy volunteers determined by MSOT does not differ when the follow-up 
is measured after 2 weeks. 
 
The quantitative proportion of collagen signal in muscles of healthy volunteers 
determined by MSOT does not differ when the follow-up is measured after 2 weeks. 
 
The quantitative proportion of hemo/myoglobin signal in muscles of healthy volunteers 
determined by MSOT does not differ within a muscle of healthy volunteers. 
 
The quantitative proportion of oxygenated/deoxygenated hemo/myoglobin signal 
determined by MSOT does not differ within one muscle of healthy volunteers. 
 
The quantitative fraction of collagen signal determined by MSOT does not differ within 
one muscle of healthy subjects. 
 
The quantitative amount of hemo/myoglobin determined by MSOT does not differ 
between two measurements by the same examiner. 
 
The quantitative amount of oxygenated/deoxygenated hemo/myoglobin determined by 
the MSOT does not differ between two measurements by the same examiner. 
 
The quantitative amount of collagen determined by the MSOT does not differ between 
two measurements by the same examiner. 
 
The quantitative amount of hemo-/myoglobin determined by the MSOT does not differ 
between measurements made by two different examiners. 

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 
The quantitative amount of oxygenated/deoxygenated hemo-/myoglobin determined by 
the MSOT does not differ between measurements performed by two different 
examiners. 
 
The quantitative amount of collagen determined by the MSOT does not differ between 
measurements made by two different examiners. 
 
Primary objective: 
 
Comparison of the quantitative proportion of hemo/myoglobin and collagen signal in 
muscle tissue of healthy subjects before and after physical exercise (6-MWT) 
determined by MSOT, as well as over time. 
  
Secondary objectives: 
 
Comparison of the quantitative amount of hemo/myoglobin and collagen signal 
determined by MSOT in muscle tissue of healthy volunteers at timepoint 1 and 
timepoint 2 after two weeks 
 
Comparison 
of 
the 
quantitative 
proportion 
of 
oxygenated/deoxygenated 
hemo/myoglobin signal in muscle tissue of healthy volunteers at timepoint 1 and 
timepoint 2 after two weeks 
 
Comparison of the MSOT-determined quantitative amount of hemo/myoglobin and 
collagen signal at three muscle positions of two muscles (quadriceps femoris and 
gastrocnemius)  
 
Comparison 
of 
the 
quantitative 
proportion 
of 
oxygenated/deoxygenated 
hemo/myoglobin signal determined by MSOT in three muscle positions of two muscles 
(quadriceps femoris and gastrocnemius) 
 
Comparison of the quantitative proportion of hemo/myoglobin and collagen signal 
determined by MSOT in muscle tissue of healthy female and male volunteers  
 
Comparison of the quantitative proportion of oxygenated and deoxygenated 
hemo/myoglobin signals determined by MSOT in muscle tissue of healthy female and 
male volunteers  
 
Correlation of the hemo/myoglobin and collagen signal determined with MSOT with the 
6-MWT 
 
Correlation of the oxygenated/deoxygenated hemo/myoglobin signal determined by 
MSOT with the 6-MWT 
 
Comparison of the quantitative amount of hemo/myoglobin and collagen signal 
determined by MSOT between two independent examiners 

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 
Comparison of the quantitative amount of hemo/myoglobin and collagen signal 
determined by MSOT between two images of one examiner 
 
 
Study type 
Since no data exists to date to support the hypothesis of this study, this is an exploratory study. 
 
 
 

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7. Target parameters 
All measurements with MSOT are performed at three muscle positions of the proximal and 
distal lower extremity muscles in lateral comparison (right - left, leg proximal: Musculus 
quadriceps, distal: Musculus gastrocnemius) by two different examiners, in healthy subjects, 
at two different timepoints. 
 
Primary target: 
Quantitative hemo/myoglobin and collagen signal (in arbitrary units) 
This target is measured non-invasively by MSOT.  
 
 
Secondary targets: 
Quantitative lipid signal (in arbitrary units) 
Muscle oxygenation (in arbitrary units) 
This target is measured non-invasively by MSOT.  
 
6-minute walk test (6-MWT) 
This target value is clinically determined at presentation. 
 
Age 
Gender 
Weight 
Skin colour 
Ethnic background 
Underlying diseases 
Current medication 
 
This information is collected for the study at presentation.  
 
 

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8. Study design 
Monocentric / multicentric 
This is a monocentric study with prospective data acquisition (Investigator Initiated Trial, IIT).  
Study arms: intervention/control 
Interventions are not planned. A comparison is made between healthy volunteers. The study 
procedure is identical for all test subjects. 
Randomization 
Randomization is not planned. These is no allocation to groups.  
Blinding 
Blinding for the study is not possible due to the study design. Blinding is performed during the 
measurement and analysis of the data. Blinding of the test subjects is not necessary. 
 
 
 

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Graphical presentation of study design 
 
 

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9. Study population 
Inclusion and exclusion criteria 
Inclusion criteria: 
Healthy volunteers: 
 
Adult (>18 years of age) participants 
 
Exclusion criteria: 
Participants: 
 
anamnestic or other signs of myopathy  
 
pregnancy  
 
Tattoo on skin to be examined  
 
missing consent form 
 
Participant number 
As this is a pilot study, it is not possible to calculate the exact number of cases. It is planned 
to examine a total of 10 healthy volunteers. 
 
Recruitment routes and measures  
Healthy volunteers will be informed about the possibility to participate in the study. If they are 
willing to participate, they will be fully informed about objectives and methods (especially about 
the scientific/explorative character of the study), benefits and risks and the revocability of their 
participation in the study. 
 
 

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10. 
Study course 
Procedure for informing about and obtaining consent 
Participants can only be included in the study after a written declaration of consent has been 
given. The written declaration of consent requires that the volunteers are informed orally and 
in written form about the objectives and methods (including the scientific-explorative character 
of the study), benefits and risks as well as the revocability of their participation in the study. 
The information is provided by means of comprehensible proband information sheets. By 
giving their written consent, the volunteers declare that they agree to the collection and storage 
of data relevant to the study and their verification by monitoring or authorities. It must be clearly 
communicated to the study participant that a withdrawal of consent is possible at any time and 
without any disadvantage. Furthermore, all study participants/test persons are informed that 
this study is a purely scientific study without any current diagnostic or therapeutic benefit. 
The original of the consent form is kept in the study folder at the study site. The volunteer 
receives a copy of the patient information and consent form. The proband information and the 
consent form can be found in the appendix of this study protocol. 
Measurements 
After informing of the subjects, all study participants are 
imaged by MSOT at 3 positions of 2 anatomical muscle 
regions on both sides (right and left): upper/lower leg, on 
predefined muscle groups (leg proximal: Musculus 
quadriceps, 
distal: 
Musculus 
gastrocnemius). 
The 
examination is performed by two independent examiners 
directly one after the other. The examination is performed 
analogous to sonography over the corresponding skin 
layers without further invasive procedures. The anatomical 
region can be localized by means of built-in B-scan 
sonography; the corresponding optoacoustic signals can be recorded in parallel. Each 
examiner performs two repetitive scans per muscle position. The duration per anatomical 
region is limited to 5 minutes; this corresponds to a maximum of 20 minutes for both lower 
extremities. The subjects can remain in a relaxed body position during the examination; 
assistance in the form of breathing manoeuvres or similar is not necessary. Subsequently, a 
6-minute walk test is performed to test muscle activation. Afterwards another examination with 
MSOT is carried out analogous to the above mentioned procedure. The same procedure is 
repeated after 14 days. 
 

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Recording of target parameters 
 
Non-invasive 
in-vivo 
measurement 
of 
hemo/myoglobin, 
collagen 
and 
lipid 
content/signal and oxygenation by MSOT 
 
Assessment of the 6-minute walk test 
 
Determination of routine data (height, weight, underlying diseases, medication) 
 
Time schedule and study duration for the individual volunteer:
:  
 
 
 
 
 

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The duration of the study participation is about 125 minutes for the individual volunteer. At 
timepoint 1 the duration of study participation is 75 minutes. Approximately 20 minutes are 
allotted for the education of study participants, approximately 5 minutes for the collection of 
routine clinical data, 10 minutes for clinical (routine) testing using 6-MWT, and 40 minutes for 
the actual examination. At timepoint 2, participation time for the study is about 50 minutes. The 
clinical (routine) test using 6-MWT takes 10 minutes, and 40 minutes for the actual 
examination. 
 
Total study duration 
Depending on the number of patients, the expected total duration of the study until the inclusion 
of the last patient is approximately 6 months. 
 

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11. 
Risk-benefit analysis 
All study related risks 
Based on the classification criteria for medical devices (Directive 93/42/EEC, Annex IX), the 
optoacoustic system of iThera Medical corresponds to Class IIa: 
 
Active diagnostic device 
 
non-invasive 
 
Temporary use (<60 min) 
No CE certification is available for this research device (current type designation according to 
imprint: Acuity Echo). A conformity assessment procedure in the sense of the MPG is not 
intended or planned by the manufacturer at the present time. It is therefore a purely scientific 
pilot study. There is no dependency on the manufacturer, all diagnostic and analytical 
procedures are available to the study directors on site.  
 
Adherence to energy levels 
The laser safety and maximum permitted radiation dose for irradiation with laser pulses is 
regulated in the laser standards ANSI and IEC 60825. The MSOT system meets these 
standards and therefore remains below the MPE (maximum permissible exposure) limits for 
skin irradiation and is therefore considered safe. 
 
Temperature increases due to MSOT in tissue 
Optoacoustic imaging does not result in any significant temperature increase in the tissue. The 
absorption of a laser pulse in the tissue results in a local transient temperature increase of a 
few millikelvin. Depending on the duration of the examination and the skin type of the patient, 
temperature increases occur typically in the range of less than one degree Kelvin. 
 
Histological changes in tissue 
Histological changes in the target tissue and surrounding structures are neither expected nor 
have they been observed in previous preclinical and clinical studies. 
 
Slight, reversible redness or warming might occur in very sensitive skin.  
 
Such side effects are to be noticed at any time by the test person or doctor; the examination 
can then be interrupted or aborted. In any case, no irreversible damage is to be expected. 
 
In general, the near infrared light used in the MSOT can lead to retinal damage if the eye is 
irradiated. In order to prevent this, test participants and examiners will wear appropriate laser 
safety glasses during the examination. 

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Since the data obtained is not used to interpret diagnostic results, there is no risk of possible 
misdiagnosis or incorrect display of data in this exploratory pilot study. 
 
No other risks exist for this study, nor have we described any risks based on our own 
preliminary data. 
 
Benefits associated with the study 
The data obtained in this study can provide important information about the changes of 
quantitative MSOT signal in muscles due to physical exercise, as well as over time. The 
quantifiable differences could be important for future planning of further studies and the 
avoidance of errors in the study design. Additionally, first longitudinal data are essential to 
design appropriate studies.  
 
Termination criteria 
Termination criteria for the individual participant: 
Participation in the study is terminated if there is noticeable warming or reddening of the skin. 
The examination time per anatomical region is limited to 5 minutes, making these events highly 
unlikely. 
Due to the short duration of the study participation, no other discontinuation criteria are 
planned. 
Termination criteria for the whole study:  
A termination of the entire study is not planned. 
 
Statement on medical justifiability  
Given to previous experience, especially with children, the risk of undesirable events is 
considered as extremely low.  
No serious incident has been reported so far, neither at our site - nor in the literature. The 
majority of reported (foreseeable) problems are related either to the use of ultrasound gel for 
examination or the need to wear eye protection. The use of filter glass also explains the 
reported red visibility. This phenomenon was reversible within seconds. Table 1 shows the 
reported events from our study (MSOT_DMD, 67_18 B). 

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Table 1 – Adverse events   
 
Only measurements of extremities and no central organs are examined in this study - this leads 
to a further significant reduction of a possible residual risk. Particularly in light of the 
background of completely new therapeutic approaches, we hope that this method will provide 
us with a child-friendly diagnostic tool for the treatment of complex muscular diseases. 
 
 
 
 
Muscular dystrophy 
Duchenne N=10 
Healthy volunteer 
N=10 
Reversible adverse events- no.(%) 
 
 
     Pressure of safety goggles 
2 (20%) 
 
     Coolness of Ultrasound-gel 
 
4 (40%) 
     Red cast view 
 
1 (10%) 
Serious adverse events- no.(%) 
0 (0%) 
0 (0%) 

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12. 
Biometrics 
Explorative study: explanation of the statistical methodology, justification of the 
selected number of cases  
 
Case number calculation: 
As this is a pilot study and no information on the expected differences between the different 
groups is available yet, no case number calculation was performed. The number of cases given 
represents an estimate.  
 
Statistical methods: 
The data are given as mean value with standard deviation. Correlations are specified with the 
parametric Pearson correlation coefficient (R). According to the distribution between timepoint 
1 and timepoint 2, the differences of the mean values are statically examined with a parametric 
T-test. In all analyses, an error level of <0.05 is considered statically significant. 
 
 

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13. 
Data management und and data protection 
Data acquisition and storage  
All raw data, such as patient files, are source documents. Their availability is ensured for 
routine monitoring. The participation of the individual patients or test persons in the study is 
documented. The study leader maintains an independent list for the identification of the 
participating patients. This list containes the names and date of birth as well as the date of 
examination and pseudonymisation codes of the patients and subjects. The study leader is 
responsible for the quality of data collection and storage. The data storage (complete data) 
takes place on computers or specially designed network drives of the University Hospital 
Erlangen. The raw imaging data (no patient-related data) are stored on dedicated servers of 
iThera Medical GmbH. 
Pseudonymisation 
Prior to a scientific analysis of the materials and data of this study, all information will be 
pseudonymized according to the guidelines of the Federal Data Protection Act. 
Data transfer 
A transfer of the data or biological material is not foreseen in this study and will not take place. 
The study results can be published anonymously, but it will not be possible to draw conclusions 
about the identity of the participating persons. The data is stored for 10 years and then 
destroyed.  
Revocation, data deletion 
If the declaration of consent is revoked, data collected up to this point can be taken into 
account. The patient has the right to demand that the data be destroyed, provided that legal 
provisions do not prevent such destruction. 
 
 

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14. 
Handling of biomaterials  
No biomaterials are obtained.  
15. 
Insurance 
The participants of the study are insured via the group contract of the CCS Erlangen. 

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16. 
Signatures 
 
 
 
Dr. med. Ferdinand Knieling 
Study director 
Dr. med. Adrian Regensburger 
Study directors 
 
 
 
 
 
Prof. Dr. Regina Trollmann 
Study director 
 
 
 
 
 
Prof. Dr. med. Maximilian Waldner 
Technical implementation 
 
 
 
 
 
Prof. Dr. h.c. Dr. med. W. Rascher 
Clinic director