A Comparison of Serum and Urine N-telopeptide Marker
Conditions: Osteoporosis, Fractures, Bone
Sponsor: The Cleveland Clinic

Title: A comparison of Serum and Urine Bone Marker    
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Project Name 
A comparison of serum and urine N-telopeptide markers 
Protocol Title 
A comparison of Serum and Urine Bone Marker    
Investigators 
Lea El-Hage, James Bena, Jessica Colon-Franco, Leila 
Khan 
 
Version Date: 
September 25th, 
2021 
Version #: 01 
 
Grant obtained from Cleveland Clinic Foundation - 
Department of Endocrinology (from EMI institution) 
NCT # 
NCT05326815 
 
 
Name 
Title, Function 
Signature 
Prepared 
by: 
Lea El Hage 
Principle 
Investigator  
Reviewed / 
Approved 
by: 
*** 
 
 
 

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TABLE OF CONTENTS 
1.0 
INVESTIGATOR’S RESPONSIBILITIES ............................................................. 3 
2.0 
INVESTIGATOR’S AGREEMENT ....................................................................... 4 
3.0 
GLOSSARY ........................................................................................................ 5 
4.0 
OBJECTIVE ........................................................................................................ 6 
5.0 
OVERVIEW ......................................................................................................... 6 
 
Background.................................................................................................. 6 
 
Study Site ..................................................................................................... 7 
 
Overall Study Design .................................................................................. 7 
 
Subject Enrollment ...................................................................................... 7 
 
Inclusion and Exclusion Criteria ............................................................... 7 
 
Required Information .................................................................................. 8 
 
Sample Collection ....................................................................................... 9 
 
Sample Handling ......................................................................................... 9 
5.8.1 
Stability 9 
 
Standard of Care Assessments ................................................................. 9 
 Statistical Analyses ..................................................................................... 9 
 Sample Size Justification ......................................................................... 10 
6.0 
DATA MANAGEMENT ...................................................................................... 10 
 
Data Management ...................................................................................... 11 
 
Data Clarification ....................................................................................... 11 
7.0 
ADVERSE EVENT REPORTING ...................................................................... 11 
 
Date of Awareness .................................................................................... 11 
8.0 
ADMINISTRATION............................................................................................ 11 
 
Ethics Committee or Institutional Review Board Review ..................... 11 
 
Investigator Delegation of Responsibilities ........................................... 12 
 
Monitoring and Auditing Plan ....................... Error! Bookmark not defined. 
 
Disposition of Data .................................................................................... 12 
 
Record Retention ...................................................................................... 12 
 
Protocol Amendments .............................................................................. 12 
 
Deviations from the Agreed Protocol ...................................................... 12 
9.0 
REFERENCES .................................................................................................. 12 
 
 
 
Tables and Images: 
Image 1: Fisher’s Z test of the Pearson correlation ............. Error! Bookmark not 
defined. 
Table 1: Budget distribution outline .................... Error! Bookmark not defined.1 
 
 
 
 
 
 

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1.0 INVESTIGATOR’S RESPONSIBILITIES 
 
The Investigator is responsible for the data collection and the reporting of the clinical study 
information. The Investigator is also responsible for ensuring that the clinical study is 
conducted in compliance with all requirements of the clinical study protocol and all 
requirements as determined by the Institutional Review Board (IRB)/Independent Ethics 
Committee (IEC).  
 

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2.0 INVESTIGATOR’S AGREEMENT 
I, the investigator, acknowledge that I have read and understood this protocol.  I agree to 
conduct and supervise this study according to Good Clinical Practices, applicable FDA 
regulations for protecting the rights, safety, and welfare of subjects under my care, and 
for the control of devices under investigation.  
I agree to start the study only when appropriate institutional review board approval is 
obtained, if applicable.  I will maintain accurate, complete, and current records relating to 
my participation in the study. 
 
Investigator Signatures:  
 
 
 
 
 
 
Investigator Name  
(Please Print) 
 
Signature 
 
Date 
 
 
 
 
 
 
 
 
 
 

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3.0  GLOSSARY 
CCF 
Cleveland Clinic Foundation 
DEXA  
FRAX 
Dual-energy X-ray absorptiometry  
Fracture Risk Assessment Tool 
GFR 
Glomerular filtration rate 
IEC 
Independent Ethics Committee 
IRB 
Institutional Review Board 
PI 
Principle Investigator 
PTH 
Parathyroid hormone 
RC 
Research coordinator 
ROC 
Receiver Operating Characteristic 
SD 
Standard deviation 
SST 
Serum Separator Tube 
 
 
 
 

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4.0   OBJECTIVE 
This enrollment protocol is an effort to evaluate the correlation between serum and 
urine values of the bone marker of interest to help establish accuracy of serum 
values compared to urine. 
 
Data collected in this clinical study may also be used for scientific publication 
purposes, authored by the study investigators or by scientists collaborating with 
the study investigator(s), for the advancement of scientific knowledge on bone 
diseases.   
5.0   OVERVIEW 
 
 
 Background 
Osteoporosis is the most common metabolic disorder in the United States and it 
is estimated that approximately 19% of women and 4% of men over the age 50 
years have underlying disease. This number is expected to continue to rise. A 
silent illness at first, once presenting with fractures, it can lead to increased 
morbidity, mortality, and decreased quality of life. It carries large financial and 
societal burdens. Direct annual medical costs are estimated to be approximately 
17 to 20 billion dollars in the United States alone [1]. Therefore, it is important to 
accurately identify those at high risk.  
 
The present gold standard to diagnose osteoporosis is the Dual-energy X-ray 
absorptiometry (DEXA scan) [2] with a diagnosis based on a T-score of -2.5 SD or 
below in those without history of fragility fracture. However, many individuals 
fracture despite having normal or only mildly reduced scores. There are also 
several barriers within the DEXA technology including accessibility, cost, accurate 
reference ranges for age and demographic groups that result in missing large 
groups of people at risk for osteoporosis. The Fracture Risk Assessment Tool 
(FRAX score) is one tool that identifies those at higher risk of fracture that may 
benefit from therapy [3]. It was designed and released in 2008 and has been a 
great asset in clinical practice in stratifying risk and guiding management of 
osteopenic patients. However, FRAX may miss many individuals that may benefit 
from therapy due to its limited inclusion criteria [4]. 
   
Bone markers have been shown to predict fracture risk in postmenopausal women 
independent of bone mineral density and may help identify high risk 
individuals. Amino-terminal cross-linking telopeptides of type I collagen (NTX) 
reflects osteoclastic bone resorption. NTX can be measured in both the serum and 
in urine.  
 
The accuracy of the serum NTX is unclear. It may be less sensitive than urine NTX 
in detecting bone density changes [5]. The urine NTX overcomes circadian rhythm 
changes to bone density and is less sensitive to dietary collagen intake [6,7]. At 
present, urine markers need to be checked as a second void of the day which may 
be cumbersome for patients. Serum levels drawn with other bone labs would be 

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easier to obtain than second void urine collections. We would like to evaluate the 
correlation between serum and urine NTX in patients with osteopenia with no prior 
history of osteoporotic treatment. If the urine and serum markers are equivalent 
methods, serum levels would be preferred to identify high risk patients at risk of 
disease due to ease of collection.   
 Study Site 
This is a prospective blood and urine collection study under the supervision of 
clinical researchers. Subjects will be enrolled at the CCF  
 Overall Study Design 
This is a prospective specimen collection cohort study to evaluate the correlation 
between serum and urine values of the bone marker of interest, and their 
association with baseline DEXA scan measures and fracture risk within 6 months.  
 
Study samples will be obtained longitudinally. One collection of both serum and 
urine collection will be obtained. The urine will be collected as second void of day 
and at the same time the blood collection is drawn. Study will continue for a period 
or 1 year, with plan to enroll around 40 subjects.   
 Subject Enrollment 
Subjects will be enrolled prospectively, under Institutional Review Board (IRB) 
approval and patient informed consent. Enrollment will include patients diagnosed 
with osteopenia on DEXA scan who have not been on any medical therapy in the 
past. Recruitment will occur via phone, mail, virtual or in-office visit. Subjects will 
be consented in an office visit. Subjects will be identified either during an in-office 
visit, referral by colleague or via ICD code EPIC inquiry. 
 Inclusion and Exclusion Criteria 
Subject Inclusion criteria 
• Consent to participate in the study 
• Participants limited to subjects in the CCF 
• Age between and inclusive of 18 and 85 years of age  
• No gender exclusion 
• Patients diagnosed with Osteopenia on DEXA scan who have not been on 
any medical therapy in the past 
• Presence of normal vitamin D levels, kidney function, and parathyroid 
hormone levels (per our reference ranges) 
 
Sample Inclusion Criteria 

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• Serum sample collected in Serum Separator Tubes (SST) Gold tube and 
urine sample collected in clean container 
• Samples to be collected only at a CCF Laboratory based on standard of 
care for the CCF 
• Samples to be collected at the same time of day. Urine NTX will be 
collected as the second void of the day 
 
Subject Exclusion criteria 
• Prior history of medical therapy for osteopenia  
• Prior radiation therapy 
• Prior history of bone fracture 
• History of high risk medication associated with increased risk of fracture 
• Presence of abnormal vitamin D levels, kidney function, and parathyroid 
hormone 
• Pregnancy status (verbal) 
• Those with medical co-morbidities that increase the risk of fracture will be 
excluded and these include but are not limited to: rheumatoid arthritis, 
ankylosing 
spondylitis, 
psoriatic 
arthritis, 
diabetes 
mellitus, 
hyperparathyroidism, chronic kidney disease, pituitary disease, multiple 
myeloma, 
leukemia, 
lymphoma, 
thalassemia 
major, 
HIV/AIDS, 
malabsorption, 
Inflammatory bowel disease, 
chronic 
obstructive 
pulmonary disease, hypogonadism, chronic liver disease, untreated 
hyperthyroidism, and those with chronic immobility 
 
 
Sample Exclusion Criteria 
• Grossly hemolyzed (red) or turbid samples 
• Samples with visually detected microbial contamination 
• Sample not handled or stored as per sample handling instructions, which 
will be provided to the study sites at the time of the study initiation visit 
• Urine sample that is not collected as second void of the day  
 Required Information 
This section lists the main information that will be collected for each subject by the 
Principal Investigator (or designee).  Data will be captured in RedCap and will 
adhere to the data management plan as outlined in section 6.0. 
 
At enrollment: 
• Demographics 
• Age and Gender 
• Date of osteopenia diagnosis 
• Bone mineral density and T-score values of latest DEXA scan  
• Medical history; including history of any relevant concomitant 
conditions per CRF 

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• Date and time of blood draw and urine collection 
• Calcium supplementation, duration and dosing 
• Vitamin D supplementation, duration and dosing  
• Laboratory such as: 
o Vitamin D level 
o PTH level 
o Calcium level 
o Alkaline phosphatase level 
o Creatinine level 
o GFR 
 
 Sample Collection  
• Consent subjects prior to blood draw and urine collection 
• Laboratory tests will only be collected at CCF laboratory facilities per CCF 
standard of care 
• The serial number labels should be affixed to each collection tube or 
container prior to the collection 
• Patient samples will be serum from SST tubes Gold tubes and urine test 
from clean container. CPT code 82523 
• Standard venipuncture must be done in accordance with institutional 
standards and requirements to collect whole blood 
• A 2 mL SST tube of whole blood will be collected at study enrollment onset  
• Standard urine collection will be done in accordance with institutional 
standards. The urine collection will be second void of the day  
• A 5 mL clean container of second void of the day urine will be collected at 
study enrollment onset  
 
 Sample Handling 
Will be handled per CCF laboratory standards  
 
5.8.1 Stability 
The urine and serum sample can be kept at ambient temperature for 1 day, 
if refrigerated for 7 days and if frozen for 14 days 
 
 Standard of Care Assessments 
The study will collect the results of any procedures performed as per standard of 
care.  
 
 Statistical Analyses 
Pearson or Spearman correlations will be used to measure the association 
between serum and urine markers. Similar correlation measures will be used to 
measure associations with DEXA scan results, and Comparisons of the 
correlations were performed using the cocor package in R software (version 4.0; 

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Vienna, Austria) using the methods by Meng, Rosenthal, and Rubin (1992) to 
compare the correlations and calculate 95% confidence intervals for their 
difference.  As an exploratory analysis, associations between serum and urine 
levels with fracture risk will be evaluated using two-sample t-tests and, if feasible, 
ROC analyses will be used to compare the predictive ability of each marker.  If the 
number of fractures is small, this analysis will not be performed.  If the follow-up 
through 6 months cannot be measured consistently across patients, alternate 
methods including time to event analysis may be used to account for differential 
follow-up. 
 
 Sample Size Justification  
The primary sample size calculation was based on the ability to detect a moderate 
correlation (r=0.5) between serum and urine levels.  With 40 patients, there will be 
90% power to detect moderate correlations based on a two-sided test of the 
Fisher’s Z test of the Pearson correlation (Image 1).  Power calculations were 
performed using SAS software (version 9.4; Cary, NC). (8)  
 
6.0   DATA MANAGEMENT 
The Principal Investigator (PI) is responsible for ensuring that all data are 
submitted in a timely fashion. 

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 Data Management  
We will use RedCap database, only CCF MRN as identifier, assign serial number 
to each unique patient and store data on a Cleveland Clinic Shared Drive which 
will be accessed on workstations requiring login credentials. Once data are 
collected, we will de-identify the rows and use only serial numbers. 
 
Data Clarification  
During the conduct of the study, if a question regarding the data arises, the 
Monitor and/or Delegate will work with the site to resolve the issue. All 
communications will be documented in a query via the electronic database.  
7.0   ADVERSE EVENT REPORTING 
Unanticipated Non-Serious Adverse Event Reporting: 
Unanticipated non-serious adverse events assessed by the PI as being either 
caused by, or related to, the study procedures, will also be reported using the 
Adverse Event Report form.   
 
The following adverse events are anticipated as a result of the blood collection 
required per the study procedures: pain, bruising, bleeding or infection at the site 
where the blood was collected from the subject.  Fainting may also occur as a 
result of blood collection. Because these events are anticipated, they do NOT need 
to be reported using the Adverse Event Report form as long as they are not 
assessed as serious. If assessed as serious, then they need to be reported. 
 Date of Awareness  
A test site becomes aware of an MDR reportable event when medical personnel 
who are employed by or otherwise formally affiliated with the facility acquire 
information that reasonably suggests that a reportable event has occurred.  
Medical personnel include persons who are licensed, registered, or certified to 
administer healthcare, who have received a diploma or a degree in a professional 
or scientific discipline, or who are responsible for receiving medical complaints or 
adverse event reports or who supervise such persons. 
 
8.0 ADMINISTRATION 
 Ethics Committee or Institutional Review Board Review 
It is the responsibility of the Investigator to provide the Study Monitor or designee 
with written documentation demonstrating that this study has been approved by 
the Institutional Review Board (IRB)/Independent Ethics Committee (IEC).  A copy 
of the signed documentation must be returned to the study monitor or designee. 

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 Investigator Delegation of Responsibilities 
The Investigator’s decision to delegate study related responsibilities to his study 
personnel will be recorded on the Principal Investigator Delegation of 
Responsibilities and Signature Log. 
 Disposition of Data 
All data and information obtained during this evaluation are the property of CCF 
and may be used in support of regulatory submissions. 
 Record Retention 
The Principal Investigator will ensure that study records are maintained in 
accordance with IRB/IEC  
At a minimum the following documentation will be maintained:  
• 
All correspondence with the IRB/IEC, study monitor or designee, including 
required reports 
• 
The protocol and regulatory documentation 
• 
Copies of all data  
 Protocol Amendments 
Amendments to this protocol will be signed by the Investigator, and approved or 
exempted by IRB/IEC as required, prior to implementation. 
 Deviations from the Agreed Protocol 
Deviations from the written protocol should be avoided.  If deviations occur, they 
must be reported immediately to the Study Monitor or designee and recorded in a 
Protocol Deviation Memorandum. 
 
9.0 REFERENCES 
1. Becker, D.J., M.L. Kilgore, and M.A. Morrisey, The societal burden of 
osteoporosis. Curr Rheumatol Rep, 2010. 12(3): p. 186-91.  
2. Assessment of fracture risk and its application to screening for postmenopausal 
osteoporosis. Report of a WHO Study Group. World Health Organ Tech Rep Ser, 
1994. 843: p. 1-129.  
3. Kanis, J.A., et al., FRAX and its applications to clinical practice. Bone, 2009. 
44(5): p. 734-43.  
4. Silverman, S.L. and A.D. Calderon, The utility and limitations of FRAX: A US 
perspective. Curr Osteoporos Rep, 2010. 8(4): p. 192-7. 
 5. Abe Y, Ishikawa H, Fukao A (2008) Higher efficacy of urinary bone resorption 
marker measurements in assessing response to treatment for osteoporosis in 
postmenopausal women. Tohoku J Exp Med 214:51–59 

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6. Cremers S, Garnero P (2006) Biochemical markers of bone turnover in the 
clinical development of drugs for osteoporosis and metastatic bone disease: 
potential uses and pitfalls. Drugs 66:2031–2058 
7. Clemens JD, Herrick MV, Singer FR et al (1997) Evidence that serum NTx 
(collagen-type I N-telopeptides) can act as an immunochemical marker of bone 
resorption. Clin Chem 43:2058–2063 
8. Meng XL, Rosenthal R, Rubin DB. Comparing Correlated Correlation 
Coefficients. Psychol Bull. 1992; 111:172-175.