1. Bone Disease
Diagnostic and therapeutic perspectives
February 2015, prepared for:
Prepared by: Life-Force biomedical communications

2. Contents
Bone structure & function
Bone proteins
Bone metabolism & disease
Bone disease therapies
Bone disease biomarkers
Bone biomarker response to therapy
Pre-analytical requirements for
Dx use of biomarkers

3. Bone function
• Rigid framework to protect vital organs.
• Muscle attachment, allows movement.
• Hematological: home of bone marrow.
• Central role in Calcium and Phosphate homeostasis
–85-90 % of phosphate
–99% of calcium
is stored in the bones and teeth

4. Bone content
• < 70% Bone Mineral: Ca10(PO4)6(OH)2, combine and harden
the matrix into hydroxyapatite crystals
• > 10% water
• < 20% organic
• 85% Type I collagen fibres
• non-collagenous proteins like osteopontin, osteocalcin
• proteoglycans
• growth factors like IGFs, TGF-ß, PDFGs, BMPs
(Ca10(PO4)6(OH)2)

5. Bone collagen type 1
major organic bone component
• The basic structural unit is a triple helix molecule.
• In fibres collagen molecules pack together side by side.
• Crosslinks between the chains and between the molecules
give collagen its strength.
N
N C
Pyridinium crosslink
helical
region
telopeptide
region
{
{
C

6. Bone collagen crosslinks
• Found almost exclusively in bone collagen
– PYD in cartilage, bone and ligaments; DPD in bone and dentin
– Released into circulation as a result of collagen degradation during
bone resorption

7. Bone architecture
–Cortical bone
 Comprises ~80% of bone mineral mass
 Shafts of long bones and outer surfaces of flat bones
 Mechanical strength increases with thickness
–Trabecular bone
 Comprises ~80% of bone surfaces
 Mainly at ends of long bones and inside flat bones and vertebrae
 Mechanical strength determined by number & thickness of
trabeculae, and nr. of horizontal connections
 More rapidly affected by increased bone turnover

8. Bone - Calcium & Phophate metabolism
Skeleton (storage), Gut (uptake), Kidney (secretion).
• Parathyroid hormone (PTH): Action = Ca increase in blood
• stimulates bone resorption (osteoclasts)
• stimulates renal reabsorption of Ca
• stimulates synthesis active vit D
• 1,25-dihydroxyvitamin D: Action = Ca (& Pi) increase
stimulates Ca and Pi uptake from food (through gut)
• Calcitonin (CT): Action = Ca decrease
• counteracts all PTH actions
• inhibits bone resorption (osteoclasts) and renal reabsorption
• Fibroblast Growth Factor, FGF23: Action = Pi decrease
reduces renal reabsorption of Pi, inhibits synthesis active 1,25 vit D

9. Bone turnover
• Bone resorption (osteoclasts digest type I collagen) and
bone formation (osteoblasts) is ongoing dynamic process.
• Normally balanced, 10% skeleton renewal per year.

10. Bone turnover – cellular players
• Osteoblast Cell:
• Formation, organization & mineralization of bone matrix.
• Synthesis of collagen and other bone proteins.
• Osteoclast Cell
• Responsible for mineral dissolution (by acid environment
and lysozymal enzyme action below ruffled border).
• Responsible for collagen break down (Cathepsin K and
MMP pathways).

11. Bone turnover – cellular players
• Osteocyte or star-shaped cell
• Most abundant cell in cortical bone
• Result from osteoblasts trapped in the matrix they secrete
• Networked to each other via long cytoplasmic extensions
• Function as key mechanosensors
• Destroys bone through a rapid mechanism called osteocytic
osteolysis

12. Osteocytes
star shaped cells sense mechanical loading
this inhibits sclerostin expression
which indirectly stimulates bone formation

13. Bone metabolism-biochemical overview
_

14. Bone proteins - collagenous
Bone formation Bone resorption
Collagen synthesis metabolites
Type I procollagen
N-terminal propeptide (PINP)
C-terminal propeptide (PICP or CICP)
Type I collagen
Collagen breakdown products
DPD (Deoxypyridinoline)
PYD (Pyridinoline)
NTX (Cross-linked N-terminal telopeptide)
CTX (Cross-linked C-terminal telopeptide)
ICTP (Carboxy-terminal telopeptide)
Helical peptide

15. Bone proteins non-collagenous
bone formation role/function
Protein Bone formation function
Bone specific alkaline
phosphatase (BAP)
Osteoblastic enzyme involved in bone formation
(probably plays a role in bone matrix maturation).
Osteocalcin (OC) or Bone
gla protein
Major structural protein of the bone matrix, binds
calcium and attracts osteoclasts.
Matrix gla protein (MGP)
Found in in bone, heart, kidney and lung. In bone,
its production is increased by vitamin D. Like OC a
calcium binding protein.
Osteonectin
Binds calcium and is involved in regulation
of mineralization.
Proteoglycans Role is unclear.
Bone Sialo Protein (BSP)
Major structural protein of the bone matrix. Its role
has been associated with mineral crystal formation.
Sclerostin
Synthesized by osteocytes, inhibits bone formation
by regulating osteoblast function and promoting
osteoblast apoptosis.

16. Bone proteins non-collagenous
bone resorption role/function
Protein Bone resorption function
Tartrate resistant isoenzyme
acid phosphatase (TRAP5b)
Synthesized by active osteoclasts. TRAP5b catalyzes the
formation of reactive oxygen species (ROS), that degrade bone
matrix products in resorbing osteoclasts.
Cathepsin K
Main osteoclastic protease, responsible for bulk degradation of
Type I Collagen.
Matrix Metallo Proteinases
(MMPs)
MMP-2, -9, -13, -14 participate in the degradation of the
collagenous bone matrix, resulting in epitope ICTP.
Lysozymal enzymes
Enzymes secreted by the osteoclasts, responsible for mineral
dissolution in an acid environment.
Calcitonin receptor
Osteoclastic receptor for calcitonin, an inhibitor of osteoclast
activity.
RANK(NF-κB ) Osteoclastic receptor for sRANKL
Osteopontin
Cell-binding protein, synthesized by otstoblasts, that anchors
osteoclasts to mineralised matrix.
sRANKL
Soluble Receptor Activator of Nuclear Factor (NF)-κB Ligand.
Binds to RANK and is the main stimulatory factor for the formation
of mature osteoclasts.
OPG (Osteoprotegerin)
OCIF (Osteoclast Inhibiting Factor)
OBF (Osteoclast Binding Factor)
Key factor in inhibition of osteoclast differentiation and activity.
Binds and acts as as decoy receptor for s-RANKL.

17. Bone formation stimulators & inhibitors
Protein Stimulation Inhibition
Systemic
• PTH
• PTHrP
• Sex-steroids E2,T)
• Growth hormone
• IGF-1
• Thyroid hormone
• Leptin
• Glucocorticoids
• β-2 adrenergic
• Leptin
• Serotonin
Local
• Transforming growth factor-ß
• BMP-2
• Platelet-derived growth factors
• Endothelial growth factor (EGF)
• Vascular EGF
• IGF-1
• Fibroblast growth factors (FGF-2)
• Prostaglandins
• Wnt-LRP5/6
• Sclerostin
• Noggin
• Interleukin-1ß,7
• Interferon- Ƴ
• Tumor necrosis factors
• Dickkopfs (DKK-1)

18. Bone resorption stimulators & inhibitors
Protein Stimulation Inhibition
Systemic
• PTH
• PTHrP
• 1,25 (OH2)D3
• Thyroid hormone
• β-2 adrenergic
• Calcitonin
• Sex steroids
(Estrogen, Testosterone)
Local
• RANKL
• Macrophage-CSF
• Granulocyte macrophage-CSF
• Interleukin-1,6,7,11,15,17
• Interferon-Ƴ
• Tumor necrosis factor-α (TNF-α)
• Fibroblast growth factors
• Prostaglandins
• Osteoprotegerin (OPG)
• Transforming growth factor-ß (TGF-β)
• Interferon-Ƴ
• Interleukin-4,10, 13,18
• Interleukin-1 receptor antagonist

19. Bone metabolism-biochemical overview
_

20. Bone disease
• Aging, disease or other conditions may cause imbalanced
bone resorption/formation, processes occur at different
rates.
• Osteoporosis is the main clinical bone disease (low bone
mass & structure deterioration), affects 1/3rd of women 60-
70 years, and 2/3rd aged > 80.
Men
Women
20 40 60 80
Fracture
Zone
Years
Bonemass

21. Osteoporosis pathogenesis
* Reproduced from Graham Russell, with permission.

22. Metabolic bone disorders
• Hyperparathyroidism
• Hyperthyroidism
• Renal failure
• Vitamin D disorders
• Paget’s disease
• Metastatic cancer to bone
• Nutritional rickets
• Osteomalacia
• Multiple myeloma
• Malabsorption syndrome
• Systemic lupus
erythematosus (SLE).
• Prolonged immobilization

23. Drug-induced bone disorders
• Immunosuppressive drugs
• Heparin, used in kidney dialysis
• Phenytoin for epilepsy
• Glucocorticoids (corticosteroids) for rheumatoid arthritis
• Glucocorticoids (corticosteroids) for asthma
• Aluminium intoxication
• Vitamin A intoxication
• Thiazide use.

24. Bone disease therapies - summary
• Non-pharmalogical:
– diet/supplements/exercise/prebiotics….
• Agents inhibiting resorption:
– Bi(s)phosphanates
– Hormone replacement therapy (HRT)
• Agents stimulating bone formation:
– PTH
– Growth factors
• Agents inhibiting resorption and stimulating formation
– Strontiumranelate
• Biologicals:
– Monoclonal antibodies or small biomolecules affecting
resorption or formation

25. Bone disease therapies
*A combination of prebiotic short- and long-chain inulin-type fructans enhances calcium absorption and bone mineralization in young
adolescents
Steven A Abrams, Ian J Griffin, Keli M Hawthorne, Lily Liang, Sheila K Gunn, Gretchen Darlington and Kenneth J Ellis
American Journal of Clinical Nutrition, Vol. 82, No. 2, 471-476, August 2005
Therapy Application
Non-Pharmacological approaches
Nutrition
Sufficient protein, calcium and vitamin D intake is
recommendable for all ages. A good diet shortens hospital stay
after hip fracture.
Inulin: daily consumption of a combination of prebiotic short- and
long-chain inulin-type fructans significantly increases calcium
absorption and enhances BMD during pubertal growth*
Exercise
Regular exercise reduces risk of ostoperosis and delays decrease
BMD. High-charge exercise increases BMD at hip + forearm.
Fall prevention
Smoking Cessation
Smoking is osteoporosis risk factor. Cessation may have positive
effect on BMD.
Calcium and Vitamin D
Calcium Prevention osteoporosis
Vitamin D (calitriol)
Active form of vitamin D given to post-menopausal women who
have osteoporosis in the spine.

26. Bone disease therapies
Therapy Application
Anti bone-resorption agents
Inhibit bone resorption and increase mineral density. Actions reduce loss of bone mineral, leading to
increased mineralization and mineral density. Decreased fracture rate during first 5 years. No rebuilding of
bone that is lost in osteoporosis.
Hormone replacement
Estrogen
Use for shortest time at start of menopause, prolonged use
increase breast cancer and cardiovascular risks.
Tibalone
Increases BMD effectively. However, significant increase of risk
of stroke.
Selective estrogen receptor modulators Prevent bone loss and increase BMD (Raloxifene, (Evista))
Phytoestrogen
Natural source of estrogen as found in soy. Used by women as a
“safe”alternative to HRT.
Biphosphanates
(Alendronate (Fosamax); Risedronate (Actonel),
Ibandronate, Zoledronic acid).
Prevent bone loss and increase BMD. Rigid administration is
disadvantage. Under FDA review, because of alleged risks for
long-term use by post-menopausal women
Calcitonin (Miacalcin)
Treatment of osteoporosis and Paget's disease, considered not as
effective as biphosphanates. Decreased tolerance with long-term
use (nasal spray)

27. Bone disease therapiesTherapy Application
Bone-formation stimulating agents
Sodium floride
Increases BMD, however, clinical studies showed no decrease in
vertebral fracture ratesf
Parathyroid hormone (PTH ) analogue
(Teriparatide; Forteo)
PTH increases bone turnover, and it increases formation of bone
more than resorption. Results in new bone formation; a
laying down of protein matrix and mineralization,
occurring in the previous existing matrix, but also in the
new structure. Treatment of osteoporosis. PTH increases spinal
bone density and hip bone density. The relative risk of having
moderate vertebral fracture is greatly reduced .
Growth factors
Growth hormone therapy is used (and FDA approved) in the
treatment of hypopituitarism and somatotropin deficiency of
children and adults.
Agents inhibiting bone resorption and stimulating bone formation:
Strontiumranelate (Protelos; Servier)
Registered as a prescription drug in > 70 countries for treatment
of post-menopausal osteoporosis to reduce risk of vertebral and
hip fractures. In US, not approved by the FDA.
Biologicals:
Antibody to Sclerostin (Amgen, AMG-785)
Stimulates bone formation, Increases markers of bone formation
in post-menopausal women
Sclerostin small molecule inhibitors
(OsteoGeneX Inc.)
As above
human monoclonal anti s-RANKL antibody,
inhibits bone resorption (Denosumab, AMGEN)
Approved by FDA for use in postmenopausal women with risk of
osteoporosis in June 2010

28. Diagnosis of bone disease
• Measurement of Bone Mineral Density (BMD)
at hip and/or spine is the gold standard.
• BMD reflects bone history, it is not predictive.
• Detectable changes in BMD take years to develop.
• BMD does not provide information on deterioration
of bone tissue architecture.
• Increasing number of drugs therapies available for
treatment requires use of rapid and predictive methods to
diagnose disease and to assess the efficacy of therapy.
• All biochemical players of bone metabolism are potential
biomarkers.

29. Bone disease biomarkers
• Elucidation of bone resorption and formation processes has
offered many biomarker candidates.
• Ideal bone disease biomarkers should demonstrate:
• Differences pre-and post-menopause
• Significant changes in response to treatment
• Detect changes in short time intervals (months)
• Minimal analytical variation
• Minimal within person variation
• Preferably little variation over the day
• Preferably high stability in specimen

30. Main routine biomarkers of Bone
Turnover & Calcium/Pi metabolism
BONE FORMATION
• BAP
• Osteocalcin
BONE RESORPTION
• CTX-I
• NTX-I
CALCIUM METABOLISM
• PTH
• 25 vitamin D
• 1,25 vitamin D
• Calcium
• Calcitonin
SPECIAL SITUATIONS
• TRAP5b (dialysis patients)
• Sclerostin (osteocyte function)
• OPG/sRankl (regulation of bone
turnover)
Pi METABOLISM
• FGF23

31. Therapy monitoring using biomarkers
hormone replacement therapy (HRT) - BAP
• Mean BAP decreased 32% from baseline after 12 months in HRT-
treated women, a significant difference from placebo group
HRT
Placebo
Months of Therapy
BAP
(U/L)
10
15
20
25
0 12 24 36
International Osteoporosis Foundation
(IOF) recommendation: for a 90 %
specificity to predict a positive BMD
response
(+3 %), BAP value of an individual patient
should decrease 20 to 40 % from the
baseline value.
Data on file, Metra Biosystems; source: Merck Research Laboratories.

32. Therapy monitoring using biomarkers
hormone replacement (HRT) and biphosphanate
therapy– Osteocalcin (OC)
Greenspan, S. L. et al. J
Clin Endocrinol Metab
2005;90:2762-2767

33. Therapy monitoring using biomarkers
hormone replacement therapy (HRT) - DPD
Months of Therapy
Dpd Spine BMD
BMD
% ∆
Dpd
% ∆
-1
0
1
2
3
0 3 6 9 12
-30
-20
-10
0
Hip BMD
Postmenopausal women treated with hormone replacement therapy
Rosen et al. J Clin Endocrinol Metab 1997;82:1904; Chesnut et al. Am J Med 1997;102:29

34. Alendronate
10 mg/day
Placebo
Months of Therapy
BAP
(U/L)
6
8
10
12
14
16
0 3 6 9 12
Therapy monitoring using biomarkers
biphosphanate therapy - BAP
• Mean BAP decreased 31% from baseline after 3 months in BP
treated women, reached nadir at 6 months, a 43% decrease.
International Osteoporosis Foundation
(IOF) recommendation: for a 90 %
specificity to predict a positive BMD
response(+3 %), BAP value of an
individual patient should decrease 20 to 40
% from the baseline value.
• Bettica P, Bevilacqua M, Vago T, et al. Short-term variations in bone remodeling
biochemical markers: cyclical etidronate and alendronate effects compared. J Clin
Endocrinol Metab 1997;82:3034-9

35. Therapy monitoring using biomarkers
bisphosphonate therapy – BAP, OC, NTx, PYD
Changefrombaseline(%)
-100
-75
-50
-25
0
25
50
0 3 0 3 0 1 0 1
74% 35%
57%
57%
BAP OC NTx Pyd
* response (% change) exceeds long-term intra-individual CV of placebo-treated subjects (92%
confidence level). Rosen et al. Calcif Tissue Int 1998:63:363-8
Months on Therapy

36. Therapy monitoring using biomarkers
PTH therapy - BAP
• PTH initially stimulates bone formation and later increases bone
remodeling.
Daily PTH treatment induced an increment
in spinal BMD of 6.1 %. An increase of >
30 %. BAP at 3 months had a predictive
value of more than 73 % for an increase in
spinal BMD of at least 3 %. Over 15
months of therapy, BAP values
had more than doubled (+116 % see
Figure).
Cosman F, Nieves J, Zion M, Woelfert L, Luckey M, Lindsay
R. Daily and cyclic parathyroid hormone in women receiving
alendronate. NEJM 2005; 353(6):566-75.

37. Therapy monitoring using biomarkers
PTH therapy– CTX & OC
Mean serum β-CTX increased with 180% from baseline to the 3rd month and 36%
from baseline to 1st month, and 125% from the 1st to the 3rd month. Mean
osteocalcin increased with 165% , from baseline to the 1st month with an
additional increase (11%) from the 1st to the 3rd month.
Isabel Oliveira de Sousa, Erik
Trovão Diniz, Thyciara Fontenele
Marques, Luiz Griz, Mário de
Almeida Pereira Coutinho,
Francisco Bandeira Arq Bras
Endocrinol Metab. 2010;54/2
http://www.scielo.br/pdf/abem/v54n
2/23.pdf

38. Therapy monitoring using biomarkers
PTH therapy - Sclerostin
………………….
0.00
0.20
0.40
0.60
0.80
1.00
0 5 10 15
SOST (ng/ml)
hours after PTH injection
Data on file, Tecomedical

39. Therapy monitoring using biomarkers
TRAP5b during hemodialysis
TRAP5b assay levels
are unaffected by
dialysis. In contrast
serum NTX and
osteocalcin decreased
significantly after
hemodialysis sessions
(indication for
accumulation in uremic
serum), BAP increased
slightly.
Shidara K. et al. Serum levels of
TRAP5b, a new bone resorption
marker unaffected by renal
dysfunction, as a useful marker of
cortical bone loss in hemodialysis
patients. Calcif Tissue Int. 2008
Apr;82(4):278-87. Epub 2008 Apr 18.

40. Therapy monitoring using biomarkers
TRAP5b predialysis patients with chronic kidney disease
TRAP5b in both CKD patient
groups was significantly higher
than the normal value of healthy
individuals . Authors conclude
that TRAP5b demonstrated
superiority over NTX as a bone
resorption marker.Assessment of
bone metabolism in such patients
using osteocalcin and NTX may
in fact result in overestimation of
bone turnover.
Yamada S et al. Utility of serum
tartrate-resistant acid phosphatase
(TRACP5b) as a bone resorption marker in
patients with chronic kidney disease:
independence from renal dysfunction.
Clin Endocrinol (Oxf). 2008 Aug;69(2):189-
96. Epub 2008 Jan 23.

41. Biomarker behavior in bone diseases

42. Biomarker response to various therapies
Therapy
Biomarker BAP Osteocalcin NTX-I CTX-I DPD TRAP5B Sclerostin
Hormone replacement ▼ ▼ ▼ ▼ ▼ ▼ ▼
Biphosphonates ▼ ▼ ▼ ▼ ▼ ►
PTH analogues ▲ ▲ ▲ ▲ ▲ ▼
Growth hormone ▲ ▲
Strontium ranelate ▲ ▲ ► ►
Antibody to Sclerostin
(Amgen, AMG-785)
▲ ▲ ▲►
Sclerostin small molecule
inhibitor
(OsteoGeneX Inc.)
Human monoclonal anti
s-RANKL antibody,
(Denosumab, AMGEN)
▼
Biomarker response is indicated as an increase or decrease from the baseline biomarker values (▲, increase; ▼, decrease; ►, no
response or conflicting results reported by different studies; ▲►, initial increase, then return to baseline) so the biomarker value
measured in the patient before onset of the therapy

43. Preanalytics
Critical parameters in sample collection
preceding Dx procedures.

44. Preanalytics for bone markers
• Diurnal/daily variation determines time of sample
collection
• Effect of food intake or diet determines fasting before
sample collection
• Age dependent normal values
• Sample type
• Sample stability

45. Diurnal Variation

46. Variation by age

47. Preanalytics for biomarkers
Bone Marker Sample Type Sample preparation Sample stability
Bone Formation
BAP
Serum, cell culture,
heparine plasma
Don’t treat with
EDTA or citrate because of the inhibition of the BAP enzyme
2-8 ºC for storage ≤ 5 days
-20 ºC for longer storage
Osteocalcin Intact
Serum, Heparin-
or EDTA-Plasma
Samples must be separated within 1 hour after blood collection
2-8 ºC for storage ≤ 4 hours
-20°C for storage ≤ 3 years
Osteocalcin
(1-49) and (1-43),
N-terminal & mid-regional
Serum, Plasma
Whole blood should be collected and must be allowed to clot for
minimum 30 minutes at room temperature before the serum is
separated by centrifugation (850 – 1500xg for 10 minutes). The serum
should be separated from the clot within three hours of blood collection
and transferred to a clean test tube. It is necessary taking care of the
sample collection procedure to avoid haemolysis.
Serum 2-8 ºC for storage ≤ 6 days
≤ -20 ºC for longer storage
Stable ≤ 3 freeze/thaw cycles
PINP (IDS) Serum, Plasma Samples should be separated as soon as possible after collection. 5 days (2-8°C)
Avoid repeated freeze/thaw
PICP/CICP
Serum, Heparin- and
EDTA Plasma, cell
culture
Avoid hemolysis of specimens
2-8 ºC for storage ≤ 5 days
-20 ºC for longer storage
Stable ≤ 3 freeze/thaw cycles
DKK-1 Serum, cell culture
Avoid lipemic or hemolyzed samples Before use, thaw samples at low
temperature and mix thoroughly. Samples must be diluted 1:4 prior to
use in the assay. An additional buffer and assay protocol is available
for cell culture samples.
Samples store at -20°C.
Avoid repeated freeze/thaw cycles.
BMP7 Serum

48. Preanalytics for biomarkers
Bone Marker Sample Type Sample preparation Sample stability
Bone Resorption
NTX-serum Serum, cell culture
- Collect sample in the morning or always at the same time of the day
for each individual.
2-8 ºC for storage ≤ 24 hours
-20 ºC for longer storage
Stable ≤ 3 freeze/thaw cycles
NTX-Urine Urine, cell culture
Collect second morning urine or 24 hrs urine without adding
preservatives
Room temperature ≤ 24 hours
2°-8°C for storage ≤ 72 hrs
Stable ≤ 3 freeze/thaw cycles
CTX-Serum
(Cartilaps)
Serum, plasma
Separate the serum from the cells within
3 hours after collection of blood. For optimal results it is recommended
to draw blood as fasting morning samples.
Freeze (<-20°C) samples immediately.
CTX-Urine
(Cartilaps)
Urine
Second morning void urine specimens, but any spot urine sample may
be used.
4ºC for storage ≤ 24 hours
-20 ºC for longer storage
DPD
Urine
Cell culture
Use preservative-free first or second morning urine. Collect specimens
before 10 a.m.
2-8 ºC for storage ≤ 7 days
-20°C for longer storage. Protect specimens from
exposure to light.
PYD
Urine
Cell culture
Use preservative-free first or second morning urine. In case of
longitudinal studies, urine should be collected at the same time every
day
2-8 ºC for storage ≤ 7 days
-20°C for longer storage. Protect specimens from
exposure to light.
Stable ≤ 3 freeze/thaw cycles

49. Preanalytics for biomarkers
Bone Marker Sample Type Sample preparation Sample stability
Bone Resorption
PYD-serum serum
Filter samples by adding 200 µl to a 30kMWCO Spin filter.
Centrifuge 30 minutes at 3,000-10,000 x g. Protect filtrate
from exposure to light.
2-8 ºC for storage ≤ 4 days
-20°C for longer storage.
Stable ≤ 3 freeze/thaw cycles
Helical peptide
Urine
Cell culture
Collect preservative-free urine. Collect before 10 a.m
2-8 ºC for storage ≤ 7 days
≤-20°C for longer storage
Stable ≤ 4 freeze/thaw cycles
ICTP
(Orion)
Serum recommended,
plasma also possible. Do
not use heparin or citrate
plasma samples.
2-8 ºC for storage ≤ 5 days
≤-20°C for longer storage
Avoid repeated freeze/thaw cycles.
Cathepsin K Serum, cell culture
Blood samples should be centrifuged within 90 minutes.
Prior to assay, specimens should be well shaken
4 ºC for storage ≤ 2 days
≤-20° C (preferably -70° C) for longer storage
Stable ≤ 4 freeze/thaw cycles
Sclerostin Serum, cell culture
Non-lipemic human serum
Centrifuge collected
blood samples within 4 hours
Room temperature ≤ 3 days (serum)
2-8 ºC for storage ≤ 5 days
≤-20°C for storage ≤ 2 years
≤-80°C for longer storage
Stable ≤ 3 freeze/thaw cycles
TRAP (5b) Serum or plasma (Heparin)
Room temperature ≤ 8 hours
2-8 ºC for storage ≤ 2 days
≤-20°C for storage ≤ 8 months
≤-80°C for longer storage
Stable ≤ 3 freeze/thaw cycles

50. Preanalytics for biomarkers
Bone Marker Sample Type Sample preparation Sample stability
Bone Regulation
OPG Plasma, serum
Room temperature ≤ 8 hours
4 ºC for storage ≤ 2 weeks
≤-20°C for longer storage
Serum is stable ≤ 3 freeze/thaw cycles
Heparin plasma samples are not stable for multiple
freeze/thaw cycles and should be aliquotted in
individual vials to avoid freeze/thaw damage.
sRANKL
Serum, plasma (EDTA,
Heparin), cell culture
Thaw samples at low temperature and mix them thoroughly.
Lipemic or hemolyzed samples may give erroneous results.
Store samples at -20°C. For long-term storage store
at -70 °C. Avoid repeated freeze/thaw cycles.
Myostatin
EDTA plasma, serum. Whole
blood is not suitable. Untreated
lipemic samples may produce
incorrect results.
Allow samples to come to
room temperature (18 - 26 °C) and mix gentle, avoid foam
formation.

51. Preanalytics for biomarkers
Bone Marker Sample Type Sample preparation Sample stability
Mineral
metabolism
(Ca++ & other)
PTH-intact EDTA-Plasma, Serum
EDTA plasma has been reported to demonstrate improved PTH
stability as compared to serum Collect whole blood without
anticoagulant or lavender [EDTA] tube. After allowing blood to clot, the
serum or plasma should be promptly separated, preferably in a
refrigerated centrifuge,
≤-20°C for storage if not tested same day
Calcitonin Serum
Collect whole blood without anticoagulant. After allowing blood to clot,
the serum should be promptly separated, preferably in a refrigerated
centrifuge. Avoid grossly hemolyzed or grossly lipemic samples.
≤-20°C for storage if not tested same day
FGF-23 (intact)
EDTA plasma or cell
culture
The intact FGF-23 molecule appears to be highly unstable
resulting in decreased immuno-reactivity over time. Specimen
collection and assay or storage procedures should be carried out
in an expeditious manner. A morning, 12 hour fasting sample is
recommended.
Samples should be assayed immediately or stored
frozen at -20ºC or below. Avoid repeated freezing
and thawing of specimens.
FGF-23 (C-terminal) Plasma, cell culture
Centrifuge the sample and separate the
plasma or media from the cells. Samples should be assayed
immediately or stored frozen.
≤-20° C for storage
Avoid repeated freeze-thaw cycles.
25-OH-Vitamin D Serum, cell culture
2-8 ºC for storage ≤ 24 hours
≤-20° C for longer storage
Avoid repeated freeze/thaw cycles.
1,25-(OH)2-
Vitamin D
(Immundiagnostik)
Serum, plasma
Fresh collected blood should be centrifuged within one hour. Samples
should be mixed well before assaying. Lipemic or hemolytic samples
may give erroneous results.
2-8 ºC for storage ≤ 24 hours
≤-20° C for longer storage
Avoid repeated freeze-thaw cycles.
Serum samples can be shipped at 4-8 °C (for
example with Coolpacks) and remain stable for up to
3 days
Human soluble
α-Klotho protein
Serum, EDTA-plasma.
Test samples should be measured soon after collection. Alternatively,
freeze samples and upon use, thaw the samples at a low temperature
and mix them completely before measurement.
Store frozen and do not repeat freeze/thaw cycles.