The Copenhagen PROTECT Study: Biomarkers for Length of Hospital Stay and Loss of Muscle Mass and Function in Old Medical Patients
Conditions: Sarcopenia, Muscle Loss, Length of Stay
Sponsor: Bispebjerg Hospital

 
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Study protocol containing statistical analysis plan 
 
 
 
 
The Copenhagen PROTECT study: Biomarkers for length of 
hospital stay and loss of muscle mass and function in old medical 
patients 
 
 
NCT-number: NCT04151108 
Date of upload: 26/02/2020 
 
 
 

 
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Protocol 
As humans age, there is a gradual loss of skeletal muscle mass and strength, termed 
sarcopenia (1,2). The underlying causes of sarcopenia are yet not fully elucidated but are 
thought to be multifactorial and include increased levels of systemic pro-inflammatory 
cytokines, a decrease in anabolic hormones and changes in the neuromuscular system 
(3,4). Furthermore, physical inactivity, chronic diseases, immobilisation and hospitalisation 
are known to play an important part in the development of sarcopenia (4). Notably, muscle 
strength declines more rapidly than muscle mass with aging (3) and muscle weakness in 
the elderly is prevalent and closely linked to frailty, functional decline, immobility, falls, and 
injuries in this population (5). As such, measures of muscle strength and functional 
performance have shown to be sensitive markers of frailty in combination with 
appendicular muscle mass (5,6). 
The prevalence of sarcopenia ranges from 20-30% (aged >70yrs) within the general 
community (2,7). However, the prevalence of sarcopenia in geriatric patients after an acute 
hospital admission is substantially higher, estimated at ≈50% (8). Furthermore, successive 
events of hospitalisation have been suggested to contribute to the development of 
sarcopenia, as even short periods (4-5 days) of skeletal muscle disuse are known to 
induce muscle atrophy (9,10). Mean length of hospital stay in geriatric wards due to acute 
illness or hip-fracture is typically 7 to 11 days during which the level of physical activity is 
strongly reduced (11, 12) leading to an accelerated loss of muscle mass that many older 
patients never recover from. 
Notably, a substantial part of the deterioration in functional capacity could be avoided just 
by counteracting loss of muscle mass during hospitalization. As such, we need to identify 
sensitive biological, clinical and functional biomarkers for loss of muscle mass and function 
during hospitalization to identify patients at risk of developing sarcopenia and investigate 
the association of these biomarkers with hospital length of stay, readmissions, mortality, and 
discharge to a higher level of care. 
 
Purpose of the study 
The overall aim of the present study is to identify clinical, functional and biological 
biomarkers associated with length of hospital stay in older acutely ill patients. In addition, 

 
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we will examine sensitive biomarker assays to investigate whether biomarkers assays in 
combination with clinical and functional measures have the ability to predict loss of muscle 
mass and function in patients hospitalized in a geriatric ward. Collectively, these initiatives 
will have the potential to identify patients at risk of developing sarcopenia and thus develop 
individualized treatments that counteract physical deconditioning and improve independent 
life expectancy and quality of life in older people, and lead to substantial health-care 
savings. 
The specific aims of the present project are therefore to: 
I. 
Examine whether the level of a variety of biomarkers at admission are associated 
with length of hospital stay, time to first readmission within 90 days, and time to 
mortality within 90 days in old patients, and whether the combination of clinical and 
functional measures with these biomarkers can identify patients at risk of prolonged 
hospital stay, readmissions and mortality. Primary end-point is length of stay. 
Secondary end-points are time to first readmission, time to mortality, muscle mass, 
muscle strength and frailty score (WP1). 
II. 
Establish biomarker assays for predicting muscle loss and loss of function in old 
patients hospitalized at a geriatric ward, assessing whether levels of a variety of 
biomarkers combined with clinical and functional measures are associated with 
length of hospital stay, readmissions, mortality and whether patients are discharged 
to a higher level of care. Primary end-points are the relative change in muscle 
mass, muscle strength and muscle function. Secondary end-points are length of 
stay, time to first readmission, time to mortality, frailty score and discharge to a 
higher level of care (WP2) 
Background of the project 
Physiological adaptations to ageing 
It is well established that human skeletal muscle function decays with aging, and 
sarcopenia is now generally used to describe the age-related loss of muscle mass and 
strength, which is believed to play a major role in the pathogenesis of frailty and functional 
impairment that may occur with old age (13-19). The exact mechanism behind the loss of 
muscle strength observed with aging and development of sarcopenia is not yet elucidated 

 
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(20). However, increased levels of systemic pro-inflammatory mediators such as TNF-α, 
IL-6 and CRP, a decline in anabolic hormones and changes in the neuromuscular system 
have been identified as some of the contributing factors (3,4, 21, 22, 23). Furthermore, 
physical inactivity, chronic diseases, immobilization and hospitalization are known to play a 
part in the development of sarcopenia (4). In addition, it has been demonstrated that the 
age-related muscle atrophy is followed by an increased infiltration of non-contractile 
components within the muscle tissue, such as connective tissue and intramuscular fat 
(24;25). Considering these morphological changes it is not surprising that maximal muscle 
strength is reduced as a result of aging by approximately 1.5 % per year from the sixth 
decade and forth (26). 
Sarcopenia 
Skeletal muscle tissue accounts for almost half of the human body mass and, in addition to 
its power-generating role; it is a crucial factor in maintaining homeostasis of glucose 
metabolism and as an energy reservoir in catabolic conditions. Given its central part in 
human mobility and metabolic function, any deterioration in the contractile, material, and 
metabolic properties of skeletal muscle has an extremely important effect on human health.  
 
Sarcopenia specifically refers to the skeletal muscle atrophy and loss of muscle strength 
and function often observed with aging and is a major musculoskeletal cause of the loss of 
mobility, independence, and frailty in older adults. It typically appears as a decrease in 
muscle strength concurrent with a decrease in muscle mass. Clinical sarcopenia has been 
defined in statistical terms assuming a lower normal limit of two standard deviations below 
a mean relative appendicular muscle mass referred to findings in young healthy adults (27). 
The prevalence of sarcopenia ranges from 20-30% (age > 70years) within the general 
community (2,7), however, the prevalence of sarcopenia in geriatric patients after an acute 
hospital admission is substantially higher estimated at ≈50% (8). The etiology of sarcopenia 
is complex and involves neuronal, hormonal, immunological and nutritional mechanisms. 
Furthermore, physical inactivity and immobilization is known to play an important part in the 
development of sarcopenia (28;29). Yet, muscle mass in old age is not only lost due to 
reduced anabolism and increased catabolism, but also due to a reduced capacity of muscle 

 
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regeneration as muscle stem cell activation and proliferation becomes impaired in old age 
(30).  
The knowledge surrounding the prevalence and determinants of sarcopenia in geriatric 
rehabilitation care is scarce, and it is unknown whether specific biomarkers can predict 
physical deconditioning during hospitalization. As such, the development of novel 
interventions for this group of patients is hampered by the lack of understanding of the basic 
biological mechanisms driving sarcopenia in conjunction with other age-related diseases in 
humans. The importance of sarcopenia has recently been underlined by its inclusion as a 
reportable disease in the Centers for Diseases Control and Prevention (ICD-10-CM code 
M62.84) in October 2016 (31). However, despite the serious consequences of sarcopenia 
being widely recognized, the diagnose has yet to be implemented in clinical practice in 
Denmark. Lack of knowledge and systematic assessment hinders diagnosis and treatment 
of sarcopenia and leads to physical deconditioning during hospitalization. Consequently, 
more than 50% of geriatric patients report a decline in functional capacity after discharge, 
often leading to a need for prolonged and expensive rehabilitation interventions (8).  
As successive events of hospitalisation have been suggested to contribute to the 
development of sarcopenia, and even short periods (4-5 days) of skeletal muscle disuse are 
known to induce muscle atrophy (9,10), we have defined a prolonged hospital length of stay 
as an admission lasting >96 hours. In addition, we have defined patients discharged to a 
higher level of care as patients receiving increased relief, patients with increased need for a 
caregiver, patients referred to rehabilitation or 24-hour care, or patients moving to a nursing 
home following discharge. Readmission data will be limited to readmissions in the region of 
Zealand and the Capital Region of Denmark. 
Biomarkers 
Mechanisms that regulate skeletal muscle mass are central to the understanding of 
sarcopenia and related geriatric syndromes. Recently, GDF11, a close family member to 
myostatin (33), has been measured in human blood samples (34). When GDF11 is added 
to muscle and animals, identical signaling patterns to myostatin are seen with consequent 
myotube and muscle atrophy and inhibition of muscle differentiation (32,35). High circulating 
GDF11 levels have been related to increased disease burden and elevated risk of post-
operative complications and mortality in older adults undergoing heart surgery. Notably, 

 
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patients categorized as frail based on low grip strength and gait speed as well as self-
reported activity measures, had significantly higher GDF11 levels compared to non-frail 
controls (34). The data indicate that measurements of circulating GDF11 could be a 
biomarker that reflect frailty and related to geriatric syndromes. However, translation of these 
new findings into clinical utility needs further validation in a larger cohort. 
In addition to the atrophic effects of GDF11, it is also able to induce expression of GDF15 
(36). GDF15 is present in low levels under healthy conditions but can increase during 
disease or injury and contribute to muscle wasting by suppressing appetite, resulting in 
anorexia and drastic weight loss (37). In elderly, unintentional weight loss has been 
associated with increased in hospital morbidity and increased overall mortality (38). In an 
animal study, the suppression of appetite but not the loss of skeletal muscle could be 
reversed with a GDF15-neutralizing antibody (36). As such, the combined effects of GDF11 
and GDF15 in muscle wasting during hospitalization might affect the length of hospital stay, 
readmissions and mortality.  
A decrease in hormone concentrations appears to be associated with the development of 
sarcopenia. Especially in men, the decline in testosterone levels seems to be associated 
with a decline in muscle mass and muscle strength (39). Indeed, testosterone can increase 
protein synthesis in muscle and promote muscle regeneration by activating satellite cells 
(40). Estrogen may also promote muscle repair by activation and proliferation of satellite 
cells via the estrogen receptor α and β (41, 42). In addition, a murine study showed that 
ovariectomized rats (OVX) was unable to fully regain their muscle mass following a period 
of hindlimb unloading with a subsequent period of reloading compared to OVX rats with 
estrogen supplementation (43). The protective effect of estrogen on muscle mass during 
hindlimb unloading and reloading was also confirmed in a study of male rats (44) As such, 
estrogen might attenuate the degree of disuse atrophy. 
Studies have demonstrated that ageing is accompanied by modifications of the immune 
system, specifically immunosenescence, leading to cytokine dysregulation and a chronic 
low-grade inflammation (45, 46). This has potential detrimental consequences, as research 
has shown that biomarkers of inflammation are associated with the pathology of the most 
common chronic age-related diseases including atherosclerosis (47), chronic heart failure 
(48), osteoporosis (49), type II diabetes mellitus (50), neurodegenerative disorders (51,52) 

 
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and sarcopenia (53, 54, 55). Several studies have investigated the association of 
inflammatory biomarkers with muscle mass, muscle strength and muscle function. In vitro, 
exposure of myoblasts to TNF-α inhibits muscle differentiation and causes a decrease in 
protein synthesis (56). In addition, TNF-α binding to its receptor TNFR activates the NF-κB 
pathway causing proteolysis (57). However, results regarding the effect of inflammatory 
biomarkers on muscle mass are inconsistent and lack clear evidence as to whether these 
inflammatory biomarkers are associated with sarcopenia.  
Many studies have assessed low-grade inflammation by measuring a limited number of 
inflammatory proteins, and one study showed that elderly individuals exhibiting high levels 
of pro-inflammatory cytokines also tended to show high levels of anti-inflammatory cytokines 
(58). Thus, we aim to include a variety of both pro- and anti-inflammatory markers. 
Furthermore, distinct patient populations with co-existing pathophysiological processes 
might exhibit different biomarker profiles. A recent study found a negative association 
between systemic levels of CRP and changes in muscle mass in geriatric patients in 
response to resistance training during hospitalization, indicating that patients with 
inflammation during their hospital stay may have an increased risk of deconditioning during 
hospitalization (59). 
Regardless, systemic levels of the inflammatory marker C-reactive protein (CRP), has been 
found to be predictive of both the length of hospital stay and readmissions (60, 61). Indeed, 
geriatric patients with inflammation at admission stayed on average 3 days longer than 
patients without inflammation, evaluated by CRP levels at admission (62). Despite CRP 
being a nonspecific marker of inflammation, it has also been recognized as a predictive 
marker of in-hospital mortality in elderly patients admitted to the acute ward (63).  
Recently, suPAR was established as a biomarker of inflammation and immune activation, 
and elevated levels of suPAR are believed to reflect a state of chronic inflammation (64). 
suPAR correlates with other inflammatory markers, such as TNF-α, and patients with the 
highest levels of suPAR generally have the worst prognosis (65). Elevated levels of suPAR 
have been associated with increased risk of cardiovascular disease, cancer, type II diabetes 
and premature mortality in the general population, independent of CRP levels (66). 
However, a recent study found that the prognostic capability of suPAR did not affect all-
cause mortality in patients admitted to the emergency department (67). 

 
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In a study of HIV-infected patients, suPAR was associated with low muscle mass, while IL-
6 was associated with both low muscle mass and increased fat mass in both patients and 
healthy controls (68). Thus, there seem to be distinct inflammatory processes occurring 
simultaneously with different effects on muscle mass and fat mass, respectively.  
Further validation needs to be conducted in different patient populations to utilize the 
possible prognostic value of these biomarkers, either individually or in combination with 
functional and clinical measures. 
RESEARCH PLAN 
This project includes 2 work packages (WP), of which WP1 is a prospective cohort study of 
acutely ill older patients (65+ years) referred to the hospital and will investigate the 
association of biomarkers combined with clinical and functional measures with length of 
hospital stay, time to first non-elective readmission, muscle mass, muscle strength, frailty, 
and time to mortality.  WP2 is a prospective cohort study of older patients (+65 years) 
admitted to a geriatric ward for treatment of the acute disease and rehabilitation care and 
will investigate the possible association of biomarkers with the relative change in muscle 
mass and function, frailty score, length of hospital stay, time to first non-elective 
readmission, time to mortality and discharge to a higher level of care. Readmission data will 
be limited to readmissions in the region of Zealand and the Capital Region of Denmark. 
 
WP1: Biomarkers in the acutely ill older patient  
Hypothesis: We hypothesize that combining clinical and functional measures with systemic 
biomarkers has the potential to identify older acutely ill medical patients at risk of prolonged 
(>96 hours) hospital stays, non-elective readmissions, and mortality. 
Overview of Actions: Acutely ill medical patients (age >65 years) referred to Bispebjerg-
Frederiksberg Hospital will consecutively be enrolled in the study. We will recruit 
participants during a 1-year period.  
Assessments: Blood tests, frailty (CSHA Clinical Frailty Scale), risk of pressure ulcers 
(Braden Score), sarcopenia (SARC-F), hand-grip strength, chair-rise test and body 
composition (BIA). Blood samples will be used to assess biomarkers of muscle atrophy and 
wasting (GDF-11 & GDF-15), inflammatory cytokines and acute-phase proteins, sex 
hormones and immune activation (suPAR). All assessments will be performed within the 

 
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first 24 hours of admission. Primary endpoint is length of hospital stay. Secondary endpoints 
are time to first non-elective readmission, time to mortality, muscle strength, muscle mass 
and frailty. 
WP2: Biomarkers for changes in muscle mass and function in geriatric patients  
Hypothesis: We hypothesize that combining clinical and functional measures with 
biomarkers will give us the opportunity to elucidate a composition of biomarkers associated 
with the change in muscle mass and function during hospitalization, thus having the 
potential to identify future geriatric patients at risk of physical deconditioning and prolonged 
(>96 hours) hospital stays, non-elective readmissions, mortality, and discharge to a higher 
level of care. 
Overview of Actions: Geriatric patients (age >65 years) referred to geriatric rehabilitation 
care at Bispebjerg-Frederiksberg Hospitals will be enrolled in the study. We will recruit 
participants during a 1-year period. 
Assessments: Included patients will be monitored by blood tests immediately following 
admission and at discharge. A geriatric assessment will be performed during admission 
(Barthel Index, the CSHA Clinical Frailty Scale, SARC-F, the Orientation-Memory-
Concentration test (OMC) and De Morton Mobility Index (DEMMI). Measurements of 
muscle function (e.g. hand-grip strength, gait-speed and sit-to-stand tests) and body 
composition (BIA) will be performed shortly after admission and at discharge. Blood 
samples will be used to assess biomarkers of muscle atrophy and wasting (GDF-11 & GDF-
15), inflammatory cytokines and acute-phase proteins, sex hormones and immune 
activation (suPAR). Preliminary assessments will be performed within the first 24 hours of 
admission. Primary end-points are relative change in muscle mass, muscle strength and 
muscle function. Secondary end-points are length of hospital stay, time to first non-elective 
readmission, time to mortality, frailty, and discharge to a higher level of care. 
Project significance and future perspectives  
The project includes; 1) Identifying biomarkers in acutely ill older patient groups associated 
with length of hospital stay, non-elective readmissions, muscle mass, muscle strength, 
frailty, and time to mortality within 90 days. 2) Combining biomarker measurements with 
clinical and functional measurements for predicting loss of muscle mass and function 

 
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during hospitalization in geriatric patients, and investigating the association with hospital 
length of stay, non-elective readmissions, time to mortality, frailty, and discharge to a 
higher level of care. The findings of the study will not only have implications for geriatric 
patients but has the potential to be translated across disciplines to any patient at risk of 
hospital induced muscle wasting. 
Subjects and methods 
Subjects 
Acutely ill medical patients (age > 65 yrs) for WP1 (n=1100) will be enrolled ad hoc from 
Bispebjerg-Frederiksberg Hospital. 
Geriatric patients (age > 65 yrs) for WP2 (n=200) will be enrolled ad hoc from Bispebjerg-
Frederiksberg Hospital.  
For both WP1 and WP2 we aim to include patients with a dementia diagnosis and patients 
with delirium, as this group of patients represent a large portion of the elderly patients 
admitted to the hospital. Thus, the project aims to be representative of the general elderly 
patient population. Patients with dementia will be identified through patient journals. 
Depending on the severity of dementia and delirium, it might be possible that some 
patients are unable to sign participant consent, and in these cases, we will seek participant 
consent from a close relative or guardian. Independent medical doctors who have 
knowledge of the project but are not associated with the project and are independent to 
the interests of the principal investigator will evaluate whether these subjects are able to 
participate in the study. The medical doctors designated to perform this evaluation is 
chosen based on available independent doctors on duty in the department that day. Two 
large prospective cohort studies have previously reported that dementia is independently 
associated with frailty (69,70). Another study has shown that frail patients have an 
increased risk of developing delirium and that frail patients with delirium have a reduced 
survival rate following hospitalization compared to non-frail patients with delirium (71). This 
puts this specific patient population at increased risk of adverse outcomes and muscle 
wasting during hospitalization. As such, it is essential that we increase our knowledge of 
the underlying mechanisms and predictors of muscle wasting and the predictors of longer 
hospital stays and mortality in this specific patient population so that we might help future 

 
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patients with dementia and delirium. The project entails minimal discomfort for the subjects 
and has no permanent side effects. Should the patients with delirium regain their legal 
competency during their hospital stay, they will have to sign an independent participant 
consent. 
All subjects will have blood tests performed to evaluate levels of GDF11 & GDF15, 
inflammatory cytokines and acute-phase proteins, sex hormones and immune activity 
(suPAR). In addition, all subjects will have their hand-grip strength, chair-rise ability and 
body composition (BIA) assessed. Their frailty score and risk of pressure ulcers will be 
assessed by the CHSA Clinical Frailty Scale and Braden score, respectively. The Charlson 
Comorbidity Index will be used to assess comorbidity, SARC-F will evaluate the presence 
of sarcopenia and the Orientation-Memory-Concentration test (OMC) will evaluate 
cognitive function in all subjects 
Subjects included in WP2 will have an additional geriatric assessment including the 
Barthel Index and De Morton Mobility Index (DEMMI), which are already part of standard 
clinical practice. Functional status will additionally be evaluated by a gait-speed test.  
Information from patient records and other data sources 
Prior to obtaining participant consent we will need access to the summary in patient 
records when dementia is suspected. This is solely to confirm the dementia diagnosis as 
these patients will need to be evaluated by an independent physician to determine whether 
they are able to participate in the study.   
After obtaining participant consent, direct access to information from patient records and/or 
the National Patient Registry (NPR) is necessary for project implementation and to control 
for confounders and covariates in analyses. Participants will be informed of this in the 
participant information sheet. This information is disclosed to the investigator and includes 
information regarding: 
• age, height and weight 
• smoking habits 
• results of routine bloodwork 
• admission to the intensive care unit 
• Early Warning Score (EWS) 

 
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• disease history and the number of ICD-10 discharge diagnoses 5 years prior to 
admission, to assess comorbidity 
• discharge to a higher level of care (WP2) 
• hospital length of stay 
• readmissions within 90 days in the region on Zealand and Capital region of 
Denmark 
• number of hospitalizations (acute and elective) 1 year prior to admission 
• number of medications prescribed at admission, to evaluate polypharmacy 
• results of bloodwork associated with the project 
• results from questionnaires (Barthel Index, OMC, SARC-F, DEMMI, Braden Score 
& CSFA Clinical Frailty Scale) 
Information regarding patient mortality within 90 days of admission will be extracted from 
the Danish Civil Registry System.  
The participant consent will give the investigators as well as possible supervisory 
authorities access to obtain information from patient records to view health information 
necessary for the implementation of the project, as well as required quality-control and 
monitoring.  
Processing of personal information 
All participant data are confidential, are subject to confidentiality and will be handled in 
compliance with the law on the processing of personal information. Results from 
participants will be published in scientific journals, securing the complete anonymity of 
participants. All procedures will be conducted according to “Good Clinical Practice” 
standards, regarding initiation, monitoring and reporting. All protocols will be submitted for 
approval by the local ethics committee and Danish Data Protection Agency before trial-
initiation and the project will comply with the regulations of the General Data Protection 
Regulation (GDPR) and the Data Protection Act. 
 
 
 

 
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Recruitment and participant consent 
All acutely ill old medical patients (>65 years) are included in the project. Initial contact to 
the patients will be made immediately after admission to Bispebjerg-Frederiksberg 
Hospitals, by research personnel directly associated with the project. Initial contact will 
entail informing the eligible participants, that a scientific study is being conducted and that 
they have the possibility to take part in it. Interested participants will be given the written 
participant information and we will set up a meeting immediately or within the following 
hours for the verbal information. Interested participants will be informed that they have the 
right to bring an assessor to the meeting. 
In the case of severe dementia or delirium, it might be possible that some patients are 
unable to sign participant consent, and in these cases, we will seek participant consent 
from a close relative or guardian, should the guardianship include access to signing 
participant consent in research. Independent medical doctors who have knowledge of the 
project but are not associated with the project and are independent to the interests of the 
principal investigator will evaluate whether these subjects are able to participate in the 
study, and if so, they will also sign the participant consent. The medical doctors designated 
to perform this evaluation is chosen based on available independent doctors on duty in the 
department that day. Interested participants and their relatives or guardian will be given 
the written participant information and we will set up a meeting immediately or within the 
following hours for the verbal information. The verbal information will be adjusted to fit the 
patient’s cognitive abilities. 
The verbal information will be given by research personnel directly associated with the 
project. The meeting will take place in the hospital room to ensure that the conversation is 
as undisturbed as possible, and this will give eligible participants, relatives or guardians 
time to listen and ask questions. The verbal information will be understandable without the 
use of technical terms and will be based on the written participant information including 
aim, methods, possible risks and side-effects and disclosure of personal information. 
Possible participants, relatives or guardians will be given time to consider whether they 
would like to participate in the study before signing a participant consent. Should the 
patients with delirium regain their legal competency during their hospital stay, they will 
have to sign an independent participant consent. 

 
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The appendix “Forsøgspersoners rettigheder i et sundhedsvidenskabeligt 
Forskningsprojekt” will be enclosed with the written information and reviewed during the 
verbal information. 
Following the written and verbal information, all participants will be informed of any newly 
discovered risks, side effects or any essential changes in study design. Participants will 
also be informed if the study is terminated and the reasons for termination.  
Inclusion criteria 
All acutely ill medical patients aged 65+ years admitted to Bispebjerg-Frederiksberg 
Hospital. 
Exclusion criteria 
Exclusion criteria includes being under the age of 65 years, terminal illness, droplet or 
airborne isolation, patients who do not understand Danish and patients with temporary civil 
person registration numbers (CPR).  
Statistical considerations 
The current study will recruit participants during a 1-year period to avoid any seasonal 
differences in the patient population. The acute medical unit receives approximately 10-20 
patients a day over the age of 65, giving a total of roughly 5475 patients a year. We 
estimate a participation rate of 20% due to unwillingness to participate, inability to perform 
measurements or cognitive impairments or delirium affecting their ability to sign participant 
consent. We aim to include a total of roughly 1100 participants, of which 200 patients are 
admitted to the geriatric rehabilitation care unit. 
Overall outcome analysis 
To test whether clinical, functional and biological biomarkers are associated with length of 
stay we will perform multivariate logistic regression and to test whether clinical, functional 
and biological biomarkers are associated with loss of muscle mass and function we will 
perform multivariate linear regression. To evaluate the prognostic abilities of biomarkers 
(individual, in combination and combined with clinical and functional measures) we will use 
area under the curve for receiver operating characteristics (AUROC) statistics. 

 
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Table of summary statistics will be presented with a number of baseline variables (age, 
sex, functional measures, results of blood tests, comorbidities).  
Continuous variables will be summarized with: n (non-missing sample size), mean, 
standard deviation, median, interquartile range, number of missing values. Categorical 
variables will be reported as frequency and percentages (based on non-missing sample 
size) and number of missing values.  
Continuous and ordinal variables will be tested by Wilcoxon analysis for differences 
between groups. Categorical variables will be tested with chi-square. Missing data will be 
imputed. 
A reference group of 2058 patients over the age of 65 from Bispebjerg-Frederiksberg 
University Hospital and Herlev-Gentofte Hospital had a mean age of 78.3 years and a 
mean length of stay of 5.8 days during hospitalization. 817 of these patients (39.7%) had a 
prolonged length of stay, defined as a hospitalization lasting more than 96 hours. 
With a sample size of 1100 and the assumption that approximately 40% of old medical 
patients have a prolonged hospital stay, an AUROC of 82 will have a power of 0.9 and a 
significance level of 0.05. The primary outcome will be mean length of stay. Patients will 
be grouped in either normal or extended length of stay (<96 hours) and Cox regression 
analysis will be used to compare differences in readmission and mortality. To assess the 
discriminative ability of biomarkers with regards to extended length of stay, we will use the 
area under the curve (AUC) for receiver operating characteristics (ROC) curves. AUCs for 
different ROC curves will be compared using the DeLong test. 
Methods 
Blood tests are standard clinical procedures and will be performed by trained personnel. 
For the measurement of muscle strength and function, validated tests will be used and will 
be performed by trained physiotherapists associated with the study. Measurement of total 
and regional body composition are standard methods at Bispebjerg-Frederiksberg Hospital 
and will also be performed by trained physiotherapists associated with the study. 
Questionnaires are validated and will be carried out by healthcare professionals 
associated with the study.  

 
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• Blood tests: Blood from a peripheral vein (50 ml) will be examined for inflammatory 
biomarkers, sex hormones, immune activation (suPAR) as well as biomarkers of 
muscle atrophy and muscle wasting (GDF11 and GDF15).  
• 
Maximal muscle strength: Maximal hand grip strength will be measured by a hand-held 
dynamometer.  
• Functional tests: Habitual and maximal gait speed and sit to stand test for 30 
seconds. 
• Questionnaires: A frailty score will be assessed by the CSHA Clinical Frailty Scale, 
the risk of pressure ulcers will be assessed by the Braden Scale, sarcopenia will be 
assessed by SARC-F, cognitive status by the Orientation-Memory-Concentration test 
(OMC) and the degree of multimorbidity will be assessed using the Charlson 
Comorbidity index. Participants included in WP2 will have an additional geriatric 
assessment as part of clinical practice, including the Barthel Index and the De Morton 
Mobility Index (DEMMI).  
• Anthropometry: All subjects will have their body composition calculated by 
Bioelectrical Impedance Analyses (BIA) at admission. Patients from geriatric 
rehabilitation care will have an additional BIA measurement at discharge to evaluate 
the change in body composition during hospitalization. Weight will be assessed using 
chair scales. Height will be estimated with patients lying in a supine position by a 
segmometer using knee-height measurement and age using the equation from 
Chumlea et al (72). 
 
Risks, side effects and disadvantages 
The potential discomfort from having a blood test is very brief and rarely cause a small 
blood accumulation (bruise), which will disappear within a few days. The total amount of 
blood collected is 25-50ml, which is a quite modest amount compared to the amount 
collected as a blood donor (500 ml). There are no risks or discomfort associated with 
assessments of muscle strength or muscle function or by body composition analyses by 
BIA. 
 
 

 
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Ethical considerations and dissemination of results 
All procedures will be conducted according to “Good Clinical Practice” standards, regarding 
initiation, monitoring and reporting. All protocols will be submitted for approval by the local 
ethics committee and Danish Data Protection Agency before trial-initiation and the project 
will comply with the regulations of the General Data Protection Regulation (GDPR) and the 
Data Protection Act. Findings from the project, regardless of the outcome, will be published 
in relevant peer-reviewed scientific journals and positive, negative or inconclusive results 
will be published on www.clinicaltrials.gov. Subjects will be anonymous.  
The project is expected to provide insight into the biomarkers associated with changes in 
muscle mass, strength and function in acutely ill older patients and to elucidate the role of a 
variety of biomarkers on length of hospital stay, readmissions and mortality. Each subject 
will gain knowledge regarding their physical function and body composition, and patients 
admitted to geriatric rehabilitation will gain additional knowledge regarding their muscle 
function. Furthermore, the gained insight into biomarkers of changes in muscle mass and 
function during hospitalization and their association with length of hospital stay, 
readmissions and mortality, might prove beneficial for future geriatric patients. The project 
entails minimal discomfort and no permanent side effects; thus, it is considered ethically 
sound. All subjects are covered by Bispebjerg-Frederiksberg Hospital patient insurance. 
 
The results will be available following the end of the project with a subsequent period of data 
processing. As such, individual participant results will not be available immediately following 
examinations. However, participants will be informed of individual preliminary results, to the 
desired extent.  
 
Research biobank 
It is necessary to create a research biobank for this study, as participants are recruited ad 
hoc from the hospital based on admission during a 1-year period. We will apply for approval 
from The Danish Privacy Act to create the biobank. As such, the participant consent will 
entail permission to create a biobank with biological material.   
As per the Danish Privacy Act, excess biological material must generally be destroyed 
following the end of the project.  However, we will apply for approval from the Danish Data 
Protection Agency to create a Biobank with excess material for future research. In addition, 

 
18 
we will seek permission from all subjects to store excess biological material for future 
research. The scientific ethics committee will be notified of any possible new projects. 
Excess biological material will be coded, frozen and stored at Bispebjerg hospital for the 
next 20-40 years, with anonymization of the subjects. It is important that the material is not 
destroyed as new biomarkers are constantly being discovered and further analyses into the 
complex association between various disease states and biomarkers of loss of muscle 
mass, strength and function, might increase our knowledge of the mechanisms behind 
atrophy and loss of function in elderly patient groups during hospitalization. 
 
Financial conditions 
The project is initiated by Professor Charlotte Suetta, who is also the principal investigator. 
Funding has been rewarded to the Geriatric Research Unit at Bispebjerg Hospital by the 
Novo Nordisk Foundation, and will be used for staff salaries, laboratory tests and other 
equipment in the current project. It is planned to seek additional funding in the future through 
both public and private sponsors. The principal investigator has no financial interests in the 
project and no financial association with the sponsor. No remuneration will be given to 
participants in the study, as they are all recruited directly from the hospital and therefore 
have no financial costs from participating (i.e. transportation costs). 
 
 
 
 
 
 
 
 
 
 

 
19 
 
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