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Clinical Chemistry 44: 2139-2147, 1998;
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(Clinical Chemistry. 1998;44:2139-2147.)
© 1998 American Association for Clinical Chemistry, Inc.

Enzymes and Protein Markers

Analytical and clinical performance characteristics of Tandem-MP Ostase, a new immunoassay for serum bone alkaline phosphatase

Dennis L. Broyles1, Randall G. Nielsen1, Elizabeth M. Bussett1, W. Douglas Lu1, Isaac A. Mizrahi1, Patricia A. Nunnelly1, Tram A. Ngo1, Julia Noell1, Robert H. Christenson2,3, and Barry C. Kress1,a

1 Beckman Coulter, Inc., San Diego, CA 92196.
Departments of
2 Pathology and
3 Medical and Research Technology, University of Maryland School of Medicine, Baltimore, MD 21201.
a Address correspondence to this author at: Hybritech, Inc., P.O. Box 269006, San Diego, CA 92196-9006. Fax 619-549-9357; e-mail bckress{at}beckman.com.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The performance characteristics of the Tandem®-MP Ostase® assay, a new microplate immunoassay for bone-specific alkaline phosphatase (bone ALP; EC 3.1.3.1) in human sera, are described. Bone ALP is bound to streptavidin-coated microwells by a single biotinylated anti-bone ALP monoclonal antibody. Antigen is detected by the addition of p-nitrophenyl phosphate. The assay is performed at room temperature in <90 min. Imprecision was 2.3–6.1% with a detection limit of 0.6 µg/L. Method comparison of bone ALP measurements with the Tandem-MP Ostase assay and the mass-based Tandem-R Ostase assay (n = 285) indicated regression statistics of Tandem-MP Ostase = 1.03 Tandem-R Ostase + 0.22 µg/L, Sy||x = 4.0 µg/L, r = 0.97. Serum bone ALP values in apparently healthy men and in pre- and postmenopausal women were also similar between the two Ostase assay formats. Liver ALP reactivity determined using the slope and heat inactivation methods was similar in both Ostase assays. Liver ALP reactivity ranged from 3 µg/L (heat inactivation) to 6 µg/L (slope method) per 100 U/L of liver ALP activity, whereas bone ALP reactivity was 37 µg/L per 100 U/L of bone ALP activity, indicating a liver ALP relative reactivity of 8.1–16.2%. Similar results were obtained with the Alkphase-B bone ALP immunoassay. The Tandem-MP Ostase bone ALP assay demonstrated increased concentrations of serum bone ALP in conditions where bone metabolism is increased and showed a rapid, temporal decrease in serum bone ALP in Paget disease patients on bisphosphonate therapy. In conclusion, the Tandem-MP Ostase assay for serum bone ALP is a rapid, simple, robust nonisotopic alternative to the Tandem-R Ostase immunoradiometric assay that provides an accurate and sensitive assessment of bone turnover.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Bone-specific alkaline phosphatase (bone ALP) is a constituent of the osteoblast, the cell responsible for formation of new bone during bone remodeling. Bone ALP is a tetrameric glycoprotein attached to the osteoblast cell membrane by a carboxy-terminal glycan-phosphatidyl-inositol anchor. The action of a phospholipase releases bone ALP as a homodimer from the membrane into the circulation (1). Once in circulation, the half-life of bone ALP is relatively long (1–2 days), which most likely contributes to its low diurnal and biological variability (2)(3)(4)(5). Like most serum glycoproteins, bone ALP is cleared from circulation by the liver (6).

Serum bone ALP measurements provide a sensitive and accurate assessment of bone turnover. Numerous studies support the clinical utility of this marker in various disorders in which bone metabolism is either directly or indirectly affected. These disorders include Paget disease, bone metastases, renal osteodystrophy, and osteoporosis (4)(7)(8)(9)(10)(11). In osteoporosis, a chronic disorder where bone metabolic changes are sometimes subtle, bone ALP has proven to be an effective tool in long-term patient management (4)(12)(13)(14).

The challenge to bone ALP quantification has been differentiating bone ALP from liver ALP in human serum. Bone and liver ALP, the predominant ALP isoenzymes in human serum under nonpathological conditions, are encoded by the same gene and have an identical amino acid sequence (15)(16). However, compelling evidence suggests that structural differences in posttranslation glycosylation of bone and liver ALP exist (17)(18)(19). Various techniques that exploit these carbohydrate differences have been used to quantify bone ALP in human serum, the most common of which are heat inactivation, electrophoresis, wheat germ lectin precipitation, and immunoassay.

In selected laboratories, heat inactivation, electrophoresis, and wheat germ lectin precipitation approaches to serum bone ALP quantification have proven to be reliable (20)(21)(22)(23)(24)(25)(26). However, these assays are cumbersome, technique-dependent, require sample or gel pretreatment, and consequently have not gained widespread use in clinical chemistry laboratories. More recently these limitations have been overcome with the emergence of immunoassays for serum bone ALP. Immunoassays using monoclonal antibodies that are highly specific for serum bone ALP have been developed and are rapid, easy to perform, and reproducible across laboratories (7)(27)(28)(29).

In this study, the analytical and clinical performance of the Tandem®-MP Ostase® assay, a new enzyme immunoassay (EIA) that measures serum bone ALP, are described. Our results show that this microplate-based assay is simple, robust, specific, and sensitive to bone turnover changes in bone metabolic disorders and in response to bisphosphonate therapy. A preliminary report of these results has been published (30).


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
specimen and patient selection
All serum samples were obtained with institutional review board approval or in accordance with the Helsinki Declaration of 1975, as revised in 1983. All samples were stored at -70 °C until analysis unless otherwise noted.

Serum samples from apparently healthy, ambulatory adults with no evidence of bone or endocrine disease or not taking agents known to affect bone metabolism were used to establish reference ranges. The 200 subjects who participated in the reference range study were chosen after a review of case history forms that were completed either by the participating subjects (128 subjects) or by an endocrinologist (72 subjects). The apparently healthy adults included 52 men, mean ± SD age of 41.6 ± 17.5 years, with an age range of 21–88 years; 73 premenopausal women, mean ± SD age of 35.7 ± 8.7 years, with an age range of 21–53 years; and 75 postmenopausal women, mean ± SD age of 65.2 ± 10.5 years, with an age range of 40–86 years. The apparently healthy adults included 169 Caucasians, 13 Asians, 7 Hispanics, 6 African-Americans, and 5 of other ethnic origins.

Serum samples from patients diagnosed with Paget disease were obtained from bone centers in the United States and Australia. Samples from subjects on or off bisphosphonate or calcitonin therapy were included in the methods correlation (n = 51) and liver ALP reactivity studies (n = 35), whereas only samples from subjects off therapy were included in the bone metabolism study (n = 90). Paget disease patients participating in the bisphosphonate monitoring study received intravenous pamidronate at 60 mg/week over 26 weeks. Serum samples from these patients were stored frozen up to 2 years at -20 °C before analysis.

Serum samples with increased total ALP concentrations were obtained from patients with hepatobiliary liver disease. These samples were screened by electrophoresis, and those samples with >95% liver ALP were used for the liver ALP reactivity studies.

For the bone metabolism study, serum samples were obtained from 12 men with prostate cancer bone metastases, mean ± SD age of 70.0 ± 7.5 years, with an age range of 54–82 years, and from apparently healthy children with no known medical conditions. The mean ± SD age of the male children (n = 17) was 12.0 ± 3.9 years, with an age range of 5–17 years, whereas the mean ± SD age of the female children (n = 19) was 12.1 ± 3.6 years, with an age range of 6–17 years.

tandem-mp ostase assay
Serum, calibrator, or control (50 µL) was added to streptavidin-coated microwells (Wallac). Biotinylated anti-bone ALP monoclonal antibody (100 µL) was then added to each well followed by a 1-h incubation at room temperature with rotation at 800 rpm on an orbital shaker. The microwells were then washed three times with 300 µL of a detergent solution. After the wash step, 150 µL of buffered p-nitrophenol phosphate substrate, pH 10.4, was added to each microwell, followed by a 15-min incubation at room temperature with rotation at 800 rpm on an orbital shaker. Color development was stopped by the addition of 100 µL of 1 mol/L NaOH, and the absorbance was read at 405 nm with an automated microplate reader (Molecular Devices) after subtracting a blank reading at 600–650 nm.

Calibration of the Tandem-MP Ostase assay was established to match the calibration of the Tandem-R Ostase IRMA, using bone ALP extracted from the human osteosarcoma cell line SAOS-2 as described previously (27). The Tandem-MP Ostase assay uses six calibrators at 0, 7, 15, 30, 60, and 90 µg/L of bone ALP antigen added to a solution containing bovine serum albumin. All samples, calibrators, and controls were run in duplicate unless otherwise noted. The anti-bone ALP monoclonal antibody used in the Tandem-MP Ostase assay is the same anti-bone ALP solid-phase monoclonal antibody used in the Tandem-R Ostase IRMA.

tandem-r ostase assay
Assay procedure and performance characteristics of the Tandem-R Ostase assay for serum bone ALP have been described previously (7)(27)(31). All samples, calibrators, and controls were run in duplicate, and results were expressed in µg/L. Bone ALP antigen for the calibrators was extracted from the human osteosarcoma cell line SAOS-2. The assay dynamic range is to 120 µg/L, with a detection limit of 1.0 µg/L. Within-run and between-run CVs are <7% and 8%, respectively (7)(27)(31).

alkphase-b assay
The Alkphase-B immunoassay (Metra Biosystems, Inc.) is a single monoclonal antibody-based assay that measures bone ALP in serum. Assay procedure and performance characteristics of this assay have been described previously (28)(32)(33). All samples, calibrators, and controls were run in duplicate, and results were expressed in U/L. Bone ALP antigen for the calibrators was extracted from the human osteosarcoma cell line SAOS-2. The assay dynamic range is to 140 U/L, with a detection limit of 0.7 U/L. Within-run and between-run CVs are <6% and 7%, respectively (28)(32)(33).

short-term serum bone alp stability
Eight freshly drawn serum samples from apparently healthy adults were aliquoted and stored at 25 °C, 4 °C, -20 °C, and -70 °C for up to 14 days. At designated intervals, an aliquot was brought to room temperature and assayed for bone ALP in the Tandem-MP Ostase immunoassay. The stability of separate aliquots of these samples to five repeated freeze-thaw cycles was also tested.

long-term serum bone alp stability
Quality-control serum pools were aliquoted and stored at -20 °C, -40 °C, and -70 °C for up to 303 days. In a separate study, eight freshly drawn serum samples from apparently healthy adults were aliquoted and stored at -20 °C and -70 °C for up to 349 days. At designated time intervals, an aliquot was brought to room temperature and assayed for bone ALP in the Tandem-MP Ostase, Tandem-R Ostase, and Alkphase-B immunoassays.

linearity
Serum samples containing increased concentrations of bone ALP that were within (n = 9) and beyond (n = 5) the assay dynamic range were selected to test for assay linearity. At least three dilutions in zero diluent (calibrator A) were prepared for each serum sample to span the assay range.

antigen recovery
Various quantities of a concentrated solution of bone ALP antigen extracted from SAOS-2 cells were added to human sera containing endogenous bone ALP. The volume added was <10% of the final solution volume.

detection limit
The detection limit was defined as the bone ALP concentration corresponding to the absorbance that was three standard deviations greater than the mean absorbance for 20 replicates of the zero calibrator. This was evaluated in 15 individually calibrated analytical runs. Absorbance was converted to bone ALP concentration by interpolation between the zero and 7 µg/L calibrators.

imprecision
Within-run imprecision was determined by running 20 replicates of four quality-control serum pools averaged over six separate analytical runs. Between-run imprecision was determined by assaying three quality-control serum pools in duplicate in 18 individually calibrated analytical runs using a single reagent lot across six different laboratories, five in the United States and one in Europe.

total alp activity measurement
Total ALP activity was determined using a kinetic color test at 37 °C (COBAS BIO, Roche Diagnostics Systems) with absorbance readings at 405 nm taken at 10-s intervals for 3 min, using p-nitrophenyl phosphate substrate. Between-run CVs were <6%.

gel electrophoresis
Electrophoretic separation and quantification of ALP isoenzymes were performed using the Paragon Isopal gel electrophoresis system (Beckman Coulter, Inc.) after pretreatment of the samples with neuraminidase (20). Electrophoresis was performed according to manufacturer's instructions. Briefly, 5 µL of neuraminidase-treated sample was applied to the agarose gel. Current was applied for 25 min at 150 V. The gel was then incubated with substrate for 60 min at 45 °C. After the incubation, the gel was washed and dried, and the ALP isoenzymes were quantified by densitometric scanning (Shimadzu CS-9000U Dual-Wavelength Flying-Spot Scanner).

interfering substances
Various blood components and therapeutic agents were tested for interference in the Tandem-MP Ostase assay. Each potential interferent or buffer control was added to three quality-control serum pools. The volume of interferent or control buffer added was <=10% of the final sample volume. Four replicates of each sample were assayed. The criteria for stating no interference were based on bone ALP recoveries in the test samples that were within 10% of control recoveries.

reactivity with alp isoenzymes
Reactivity with intestinal and placental ALP.
Human intestinal and placental ALP were purchased from International Enzymes. The purity of the placental ALP preparation was determined by gel electrophoresis to be >95%. The intestinal ALP preparation required pretreatment (56 °C for 20 min) to eliminate contribution from bone ALP (34). The purity of the treated intestinal ALP was determined by electrophoresis to be >95%. Total ALP activity for the placental and treated intestinal ALP isoenzyme preparations tested for reactivity in the Tandem-MP Ostase assay was 1012 U/L and 704 U/L, respectively.

Reactivity with liver ALP.
Reactivity of liver ALP in the immunoassays was determined by two methods: slope comparison and sample heat inactivation. For each method, samples were assayed using Tandem-MP Ostase, Tandem-R Ostase, Alkphase-B, and total ALP assays.

Slope comparison.
Slope comparison was performed as described previously (33). A panel of 35 serum samples averaging >95% bone ALP as determined by electrophoresis was obtained from Paget disease patients and used as a source of samples enriched in bone ALP. A panel of 43 serum samples averaging >95% liver ALP as determined by electrophoresis was obtained from hepatobiliary liver disease patients and used as a source of samples enriched in liver ALP. Total ALP values ranged from 136 to 868 U/L and from 169 to 1564 U/L for the bone ALP- and liver ALP-enriched samples, respectively. All samples containing >95% liver ALP had recoveries within the dynamic range of the immunoassays and required no dilution. Serum samples from Paget disease patients were diluted as needed. Bone ALP immunoassay results (y-axis) were plotted vs total ALP assay results (x-axis) for each sample. The ratio of the slopes of the two regression lines was used to determine the relative reactivity of liver ALP to bone ALP in each of the immunoassays.

Heat inactivation.
Five serum samples containing >95% liver ALP and with total ALP values ranging from 1075 to 2261 U/L were obtained from hepatobiliary liver disease patients. Each serum sample (2.0 mL) was incubated for 25 min at 56 °C with intermittent mixing in a temperature- controlled water bath. Aliquots were taken at 0, 10, 15, 20, and 25 min and immediately cooled on ice. Each aliquot was then brought to room temperature and assayed in the three bone ALP immunoassays and in the total ALP assay.

bone alp immunoassay correlation
The relationship between bone ALP measured with the Tandem-MP Ostase and Tandem-R Ostase immunoassays was compared in 285 serum samples obtained from apparently healthy premenopausal and postmenopausal women, apparently healthy men, and patients with Paget disease.

statistical analysis
Nonparametric Wilcoxon analysis was used to compare results obtained using the Tandem-MP Ostase and Tandem-R Ostase assays in the patient populations used for reference range analysis. Regression lines were calculated as described by Deming (35).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
analytical performance
The within-run and between-run imprecision ranged from 2.7% to 6.1% and from 2.3% to 3.9%, respectively, for four quality-control serum pools with bone ALP values ranging from 7.2 to 75.4 µg/L.

The detection limit of the Tandem-MP Ostase assay ranged from 0.26 to 0.90 µg/L over 15 determinations, with a mean ± SD of 0.61 ± 0.18 µg/L. The mean ± SD sample recovery on dilution for 14 individual samples was 104.4% ± 4.2% (range of 98.8–110.1%). The mean ± SD analytical recovery of bone ALP antigen added to 10 individual serum samples was 95% ± 3.7% (range of 88.0–102.0%). None of the blood components or drugs tested interfered with the Tandem-MP Ostase assay (Table 1 ). Reactivity of the Tandem-MP Ostase assay with placental ALP and intestinal ALP was 0 and 1 µg/L per 100 U/L total ALP, respectively.


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  		Table 1. Interfering substances.1

sample stability
Bone ALP antigen in eight individual serum samples was stable when stored up to 14 days at -20 °C, 4 °C, and 25 °C (Fig. 1 A). Antigen recovery was unaffected by five freeze/thaw cycles (data not shown).



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  		Figure 1. Serum bone ALP stability.

(A) Eight freshly drawn serum samples from apparently healthy volunteers were aliquoted and stored at 25 °C ({blacksquare}), 4 °C ({blacktriangleup}), and -20 °C ({bullet}) for up to 14 days. Bone ALP was assayed using the Tandem-MP Ostase assay. Results are reported as the mean percentage recovery of bone ALP relative to the -70 °C control. (B) Quality-control serum pools were aliquoted and stored at -20 °C, -40 °C, and -70 °C for up to 303 days. Bone ALP was assayed using the Tandem-MP Ostase, Tandem-R Ostase, and Alkphase-B immunoassays. Results are reported as the mean percentage recovery of bone ALP relative to the -70 °C control. ({bullet}- - - -{bullet}) Tandem-MP Ostase, -20 °C; ({blacktriangleup}- - - -{blacktriangleup}) Tandem-MP Ostase, -40 °C; ({bullet}——-{bullet}) Tandem-R Ostase, -20 °C; ({blacktriangleup}——-{blacktriangleup}) Tandem-R Ostase, -40 °C; ({bullet}– - –{bullet}) Alkphase-B, -20 °C; ({blacktriangleup}– - –{blacktriangleup}) Alkphase-B, -40 °C.

Bone ALP antigen in serum was also stable when stored at -40 °C for up to 303 days, as determined with the Tandem-MP and Tandem-R Ostase assays (Fig. 1BUp ). However, when stored at -20 °C, serum bone ALP instability was noted in the activity-based Tandem-MP Ostase assay after ~100 days, which continued to the last time point tested. This was observed for both the quality-control serum pools and for the eight freshly drawn serum samples obtained from healthy volunteers. Similar bone ALP instability at -20 °C was observed with the Alkphase-B assay (Fig. 1BUp ). For the mass-based Tandem-R Ostase assay, no bone ALP instability was detected for any of the storage conditions studied (Fig. 1BUp ). As expected, serum bone ALP was stable when stored at -70 °C, as determined in the three bone ALP immunoassay formats (data not shown).

reactivity with liver alp
Reactivity of the Tandem-MP Ostase assay with liver ALP was determined using two methods: slope comparison and sample heat inactivation. For the slope comparison, the ratio of the slope of the liver ALP-enriched samples (0.06 µg/U) to the slope of the bone ALP-enriched samples (0.37 µg/U) was 0.162 (0.06/0.37) or 16.2% (Fig. 2 ). For these same serum samples, the slope ratios were 16.7% for the Tandem-R Ostase assay and 14.9% for the Alkphase-B assay. Correlation coefficients for all regression lines exceeded 0.96.



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  		Figure 2. Tandem-MP Ostase bone ALP and liver ALP reactivity.

Bone ALP and liver ALP reactivities in the Tandem-MP Ostase assay were determined from the ratio of the slopes of the regression lines for serum samples enriched in bone ALP ({blacksquare}), n = 35, and in liver ALP ({diamondsuit}), n = 43. Regression line statistics were as follows: y = 0.371x - 16.6 µg/L, r = 0.99, for the bone ALP samples, and y = 0.06x + 4.4 µg/L, r = 0.96, for the liver ALP samples.

From the slope of the samples containing >95% bone ALP, the bone ALP reactivity for the Tandem-MP Ostase assay was calculated to be 37 µg/L per 100 U/L of total ALP activity. A similar ratio was observed for the Tandem-R Ostase assay (36 µg/L per 100 U/L of total ALP activity), whereas a ratio of 47 U/L bone ALP per 100 U/L of total ALP activity was obtained for the Alkphase-B assay.

In the heat inactivation study, five serum samples enriched in liver ALP were incubated for 20 min at 56 °C to preferentially inactivate endogenous bone ALP. After heat treatment, the mean ± SD Tandem-MP Ostase reactivity was 3.0 ± 0.3 µg/L per 100 U/L of total ALP activity. Identical results were obtained for the Tandem-R Ostase assay, whereas 4.7 ± 0.5 U/L per 100 U/L of total ALP activity was observed for the Alkphase-B assay. To calculate the apparent relative reactivity of liver ALP to bone ALP in the immunoassays after heat inactivation, the bone ALP reactivity was obtained from the ratio of bone ALP to total ALP for the sera from patients with Paget disease (i.e., the slopes in Fig. 2Up ). When these values were used, the apparent liver ALP to bone ALP reactivity was 8.1% (3.0 µg/L/37 µg/L), 8.3% (3.0 µg/L/36 µg/L), and 10.0% (4.7 U/L/47 U/L) for the Tandem-MP Ostase, Tandem-R Ostase, and Alkphase-B immunoassays, respectively.

assay method comparison
Regression analysis.
Regression analysis of Tandem-MP Ostase and Tandem-R Ostase bone ALP measurements in 285 serum samples yielded the following relationship: Tandem-MP Ostase = 1.03 Tandem-R Ostase 0.22 µg/L, Sy||x = 4.0 µg/L, r = 0.97 (Fig. 3 ).



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  		Figure 3. Assay method comparison.

Serum samples (n = 285) were tested in the Tandem-MP Ostase (EIA) and Tandem-R Ostase (IRMA) bone ALP immunoassays. Regression line statistics were y = 1.03 (± 0.01)x + 0.22 µg/L (± 0.27), Sy||x = 4.0 µg/L, r = 0.97. The mean ± SD bone ALP values for the Tandem-R Ostase and Tandem-MP Ostase assays were 18.6 ± 16.2 µg/L and 19.5 ± 16.7 µg/L, respectively.

Reference intervals.
Bone ALP concentrations determined by the Tandem-MP Ostase and Tandem-R Ostase assays showed no statistical differences when apparently healthy adults were compared between the two assay formats (Table 2 ). Furthermore, both assays demonstrated a significant increase in serum bone ALP concentrations in postmenopausal women relative to premenopausal women (P <0.0001).


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  		Table 2. Summary of Ostase assay format comparisons for reference populations.

bone metabolism
Serum samples from patients with Paget disease, bone metastases, and from apparently healthy children between the ages of 5 and 17 years were tested in the Tandem-MP Ostase assay. The assay results showed that bone ALP values were increased in each of the test populations compared with apparently healthy adults (Table 3 ).


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  		Table 3. Increased bone metabolism.

treatment monitoring
Serum bone ALP values in patients with Paget disease treated with the antiresorptive bisphosphonate pamidronate decreased by 31% of baseline within 2 weeks of initiating therapy and by 83% of baseline values at the 26-week time point (Fig. 4 ). A similar bone ALP response profile was obtained with the Tandem-R Ostase assay (data not shown).



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  		Figure 4. Monitoring bisphosphonate therapy in patients with Paget disease.

Serum samples from 15 patients with Paget disease on bisphosphonate therapy (60 mg/week intravenous pamidronate for 26 weeks) were assayed for bone ALP with the Tandem-MP Ostase assay. The mean ± SE percentage change from baseline is reported.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The Tandem-MP Ostase assay is a new EIA to measure serum bone ALP. This assay is similar to the previously described Tandem-R Ostase IRMA with some analytical enhancements. The EIA is performed on a microplate format at room temperature with a total assay time of 75 min. In the studies reported here, the Tandem-MP Ostase assay imprecision of 2–6% improved on the 7–8% imprecision reported for the IRMA. Furthermore, our results showed that the Tandem-MP Ostase assay had a lower detection limit (0.6 µg/L) than the Tandem-R Ostase assay (1.0 µg/L). As seen with the Tandem-R Ostase assay, the EIA exhibited excellent linearity with sample dilution and recovery of antigen added to serum. Furthermore, quantification of endogenous bone ALP was not affected after components of nondiseased blood or compounds commonly used by elderly populations or patients with metabolic bone disorders were added to serum samples (Table 1Up ).

Bone ALP measured with the Tandem-MP Ostase assay in eight freshly drawn serum samples was unaffected by short-term (14-day) storage at temperatures of -20 °C, 4 °C, and 25 °C (Fig. 1AUp ) or by five freeze-thaw cycles. These and previously published results (27)(28) suggest that serum bone ALP activity is robust to sample processing and storage procedures encountered in clinical laboratories during routine handling and testing.

We have reported previously that the stability of serum bone ALP when stored frozen at -20 °C or -70 °C extended beyond 4 years when assayed in the mass-based Tandem-R Ostase assay (14). Our stability results to 1 year in this study support these Tandem-R Ostase assay data. However, bone ALP, when detected using the activity-based Tandem-MP Ostase and Alkphase-B assays, does not show the same stability profiles as seen with the Tandem-R Ostase assay. When stored at -20 °C, serum bone ALP values decreased by an average of 10% after storage for 100 days and continued to decrease by an average of 30% from baseline after 11 months in both activity-based immunoassays (Fig. 1BUp ). No loss of bone ALP activity was observed when samples were stored at -40 °C or -70 °C. These results suggest that for long-term serum storage, temperatures of at least -40 °C, and preferably -70 °C or colder, will ensure preservation of bone ALP activity. Generally, when serum samples are stored at temperatures warmer than -70 °C, mass-based bone ALP immunoassays should be considered.

Serum liver ALP reactivity was found to be similar in the Tandem-MP Ostase, Tandem-R Ostase, and Alkphase-B immunoassays. When the slope comparison method of analysis was used, liver ALP reactivity was 15–17%, similar to previous reports using the slope or dilution methods for these assays (27)(31)(32)(33)(36)(37)(38). The slope method of determining liver ALP reactivity has the advantage of not requiring any sample pretreatment. However, a modestly increased amount of bone ALP in serum samples containing very high liver ALP concentrations is difficult to detect by electrophoresis during sample prescreening and could increase the slope of the liver ALP regression line sufficiently to overestimate liver ALP reactivity.

When the heat inactivation method was used, the apparent serum liver ALP relative reactivity was 8.1%, 8.3%, and 10.0% for the Tandem-MP Ostase, Tandem-R Ostase, and Alkphase-B assays, respectively. These results are similar to (36)(37)(38) or slightly higher than (28) heat inactivation studies reported previously for these assays. An apparent advantage of the heat inactivation method compared with the slope comparison method is that endogenous bone ALP is inactivated and, therefore, presumably only liver ALP is measured. This may account for the lower estimate of liver ALP reactivity for the heat inactivation method. However, as pointed out by Price et al. (38), the heat inactivation method assumes all serum samples have similar ALP inactivation profiles, which is not always true (34). Furthermore, the method assumes that immunoreactivity and enzymatic activity are equally sensitive to the heat treatment. Overall, these studies demonstrate that the liver ALP reactivity of these three bone ALP immunoassays is similar and sufficiently low to allow accurate and sensitive serum bone ALP quantification in the presence of modestly increased liver ALP.

Conditions of increased bone metabolism, including Paget disease, bone metastases, and growing children, were shown to have substantially increased bone ALP values relative to the adult reference range (Table 3Up ). Furthermore, the Tandem-MP Ostase assay reflected the more subtle increase in bone turnover observed at menopause, with a 33% bone ALP increase observed for postmenopausal women vs premenopausal women (Table 2Up ). This increase is consistent with previous reports for serum bone ALP and other bone turnover markers (7)(39)(40).

Serum bone ALP measurements effectively monitor the decrease in bone turnover in response to bisphosphonate treatment in Paget disease and osteoporosis (7)(8)(14)(41)(42)(43)(44)(45)(46)(47)(48)(49). In our study of bisphosphonate-treated patients with Paget disease, bone ALP decreased by 31% within 2 weeks of initiating therapy and by 83% of baseline at the end of the 26-week study period (Fig. 4Up ). A reduction of bone turnover with bisphosphonate therapy as monitored with ALP is a reliable prognostic indicator for patients with Paget disease (45)(46)(47).

The use of serum bone ALP measurements to aid physicians in the management of bone disorders is becoming established. Attributes that make this marker particularly useful include its high sensitivity to reflect sometimes subtle bone turnover changes, as seen in osteoporosis, and its relatively low diurnal and biological variability (approximately one-half the variability seen with urinary markers of bone turnover). In addition to the effectiveness of bone ALP measurements to monitor the biochemical response of bone to bisphosphonate and other therapies that effect bone metabolism, recent data suggest that there is an important association between bone ALP concentrations in postmenopausal women and the risk of future osteoporotic fractures (13).

In conclusion, the Tandem-MP Ostase assay, a nonisotopic, activity-based immunoassay on a microplate format, provides a rapid, simple, robust, and reliable alternative to the Tandem-R Ostase IRMA for the quantification of serum bone ALP. Quantification of serum bone ALP is an accurate and sensitive indicator of bone turnover (9)(40)(50) and has proven clinical utility in the management of metabolic bone disorders (51)(52).


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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