Biochemistry Lab

Bone Research Assays

The Clinical Translational Research Center Biochemistry Core Laboratory at Cincinnati Children's Hospital Medical Center provides clinical and research assays for the study of mineral and bone metabolism disorders.

The staff uses specific methods and provides the following measurement information for completing grant applications:

Parathyroid Hormone (PTH 1-84)

This intact PTH SP assay is an immunoradiometric (IRMA) method utilizing two different polyclonal antibodies that have been purified using affinity chromatography. These two antibodies are specific for two different regions of the PTH molecule.

The first antibody is specific for PTH 39-84 (C-terminus) region. It is bound to the solid phase (polystyrene beads). The second antibody is specific for the PTH 1-34 (N-terminus) region and is labeled with Iodine-125. Samples are incubated simultaneously with both antibodies. Intact PTH 1-84 contains both the 1-34 and the 39-84 amino acid sequences and is the only form of PTH that will be bound by both antibody on the bead and the antibody labeled to I-125.

Since the antibody coupled to the solid phase is specific for C-terminal and mid-region fragments as well as intact PTH, the capacity of the solid phase has been designed to accommodate very high levels of PTH. This prevents interference by extremely elevated C-terminal and mid-region fragments in unknown samples.

Following the incubation period, each bead is washed to remove any unbound labeled antibody. The radioactivity present in the remaining bound labeled antibody is then measured using the gamma counter.

Concentrations of intact PTH present in the samples are directly proportional to the radioactivity measured.

Serum is used for this assay. It is recommended that the sample be fasting. Store serum in glass or plastic 12 x 75 test tube with cap tightly sealed at -20°C or lower.

The expected normal reference range is 10 - 65 pg/mL.

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25-Hydroxyvitamin D (25-OH)

This Vitamin D-25OH RIA assay consists of a two-step procedure. The first procedure involves a rapid extraction of D-25OH and other hydroxylated metabolites from serum or plasma with acetonitrile. Following extraction, the treated sample is then assayed using an equilibrium RIA procedure. The RIA method is based on an antibody with specificity to D-25OH. The sample, antibody, and tracer are incubated for 90 minutes at 20-25°C. Phase separation is accomplished after a 20 minute incubation at 20-25°C with a second antibody-precipitating complex. A NSB/Addition buffer is added after incubation and prior to centrifugation to aid in reducing non-specific binding.

This method generates a quantitative measure of Vitamin D-25OH and other hydroxylated vitamin D metabolites in human serum or plasma for assessment of vitamin D sufficiency. The measurement of Vitamin D-25OH is becoming increasingly important in the management of patients with various disorders of calcium metabolism associated with rickets, neonatal hypocalcemia, pregnancy, nutritional and renal osteodystrophy, hypoparathyroidism, and osteoporosis.

A fasting specimen is recommended but not required. Store the serum or plasma samples at -20°C or lower. Specimens may be stored in glass or plastic vials.

The adult reference range is 9.0 - 37.6 ng/mL.

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Total Calcium (Ca)

is measured in serum and in urine by the 911 Hitachi analyzer from Roche. Calcium reacts with the o-cresolphtalein complexone in the presence of 8-hydroxyquinoline to form a purple chromophore using an endpoint assay. The serum (0.2 mL) and urine (0.5 mL) are kept in a plastic tube at -20°C.

The normal adult reference range for serum total calcium is 8.4 - 10.2 mg/dL, and in children (2 - 10 years old) is 8.8 - 10.8 mg/dL. The intra- and inter-assay coefficient of variation is less than 1%. The minimum level of detection is 0.2 mg/dL.

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Phosphorus (P)

is measured in serum and in urine by the 911 Hitachi analyzer from Roche. The inorganic phosphorus reacts with ammonium molybdate in an acid solution to form ammonium phosphomolybdate, which is quantified using a sample blank endpoint assay. The serum (0.2 mL) and urine (0.5 mL) are kept in plastic tubes at -20°C.

The normal range for serum is 2.6 - 4.5 mg/dL and in urine 0.4 - 1.3 g/24 hours (on a non-restricted diet). The laboratory results in urine are expressed in mg/dL. The intra- and inter-assay coefficient of variation is less than 1.9%. The minimum level of detection is 0.3 mg/dL.

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Bone Alkaline Phosphatase (BALP)

is measured in serum using an enzyme immunoassay (ELISA) kit from Metratm, Quidel Corporation, San Diego, CA. The kit provides a quantitative measure of bone specific alkaline phosphatase activity in serum as an indicator of osteoblastic activity. It is a microtiter strip format utilizing a monoclonal anti-BAP antibody coated on the strip to capture BAP in the sample. The enzyme activity of the captured BAP is detected with pNPP substrate.

Serum samples (no anticoagulants) should be stored at -80o C for up to 36 months. Avoid > 3 freeze/thaw cycles.

The limit of detection is 0.7 U/L. The intra- and inter-assay coefficients of variation are 5% and 6%, respectively. The reference range for pre-menopausal females age 25-44 (n=178) is 11.6-29.6 U/L, post-menopausal females age >45 (n=107) is 14.2-42.7 U/L, and for male age >25 (n=126) is 15-41.3 U/L.

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Procollagen Type I (CIPC)

or Type I C-terminal collagen propetide is measured in serum using an ELISA kit from Quidel (Metra Biosystems).

Serum (0.50 mL) is stored at 4°C for up to five days. Storage for longer periods (up to five years) should be at -70°C. Repeated freezing and thawing of specimens should be avoided.

Ethylenediaminetetraacetic acid (EDTA) or heparinized plasma will give slightly lower values. The limit of detection of the assay is 0.2 ng/mL. The lower limit of quantification is 1 ng/mL. The higher limit of quantification is 80 ng/mL. The inter-assay coefficient of variation is 11.95%. The adult reference intervals are 69 - 147 ng/mL for women and 78 - 163 ng/mL for men.

In female children the reference intervals are 136-526 ng/mL(ages 4-10 yrs);118-961 ng/mL(ages 11-13 yrs); 110-443 ng/mL (ages 14-18 yrs).

In male children the reference intervals are 149-426 ng/mL(ages 4-10 yrs);113-943 ng/mL(ages 11-13 yrs); 130-466 ng/mL (ages 14-18 yrs).

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Carboxyterminal Telopeptide of Type I Collagen (ICTP)

is measured in serum by radioimmunoassay (RIA) using a kit from DiaSorin Inc. (Stillwater, Minn.). Serum (0.3 mL) should be kept at -70°C. Thawing and freezing apparently do not interfere with the determination.

The reference range is 1.8 - 5.0 ug/L. The sensitivity of the assay is 0.5 ug/L. The intra- and inter-assay coefficients of variation are 6.2% and 7.9%, respectively. Renal insufficiency leads to elevated ICTP concentration in blood, when the glomerular filtration rate is 50 mL/min/1.73m3 or less.

The liver does not seem to be involved in the metabolism of serum ICTP antigen. Liver disease, however, may increase serum ICTP concentration due to development of osteoporosis.

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Pyrilinks-D (D-PYD) or deoxypyridinoline-crosslinks

are measured in urine by a competitive enzyme linked immunoassay (ELISA) from Quidel Corporation (Metra Biosystems Inc.) (Santa Clara, Calif.) in a microtiter plate format utilizing a monoclonal anti-deoxipyridinoline antibody coated on the plate to capture D-PYD. The minimum detection limit is 3 nM.

The determination should be carried out using preservative-free first void urine sample: twenty-four hours, two hours (fasting), or random urine sample if the D-PYD levels are greater than 3 nM.

An aliquot of 1.0 mL should be kept refrigerated (2 - 8°C) for storage of less than seven days, or frozen at -20°C or -70°C for longer storage. Do not subject sample to more than five freeze/thawing cycles. Avoid prolong exposure to light, especially direct sunlight.

Adult reference range is 2.0 - 6.0 nMD-PYD/mMCreatinine. Values may be influenced by low estrogen production, low calcium intake, low physical activity or bone diseases such as osteoporosis, Paget's, hyperparathyroidism, hyperthyroidism, and bone metastasis. The intra- and inter-assay coefficients of variation are 9.5% and 8.1%, respectively.

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Cross-linked N-telopeptide of Type I Collagen (NTx)

is measured in urine and in serum by a competitive-inhibition enzyme linked immunoassay (ELISA) from Ostex International Inc. (Seattle, Wash.) using a monoclonal antibody. For the NTx in urine, assay should be corrected for urinary dilution by urinary creatinine analysis and expressed in nanomoles of bone collagen equivalents per liter (nM BCE) per millimole creatinine per liter (mM creatinine).

The intra- and inter-assay coefficients of variation for NTx in urine are 8% and 5%, respectively. Samples should be collected as a second morning void urine specimen or a 24 hours urine specimen. Do not add preservative to the urine specimen. Hemolysis interferes with the assay.

An aliquot of 1.0 mL should be store refrigerated (2 - 8°C) for up to 72 hours. Store frozen (-20°C or below) for longer-term storage. It is recommended to collect samples for comparison at the same time of the day as the baseline specimen. The adult reference range for NTx in urine is 3 - 65 nMBCE in premenopausal population.

The intra and inter-assay coefficient of variation for NTx in serum are 4.6% and 6.9% respectively. Serum NTx samples (0.5 mL) should be stored for up to five days at 2 - 8°C or at -40°C for longer storage, and should not be freeze-thawed more than three times. The adult reference range for NTx in serum is 6.2-19.0 nMBCE (premenopausal women) and 5.4-24.2 nMBCE (male).

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Contact Us

For more information about the Clinical Translational Research Center Biochemistry Core Laboratory at Cincinnati Children's Hospital Medical Center, please contact Theresa Kenney at 513-636-2229, theresa.kenney@cchmc.org .