A Miniaturized, Self-contained, Single-use, Disposable Assay Device for the Quantitative Determination of the Bone Resorption Marker, NTx, in Urine

^a author for correspondence: fax 408-524-2252, e-mail jblatt{at}metrika.com

We have developed an enabling technology called MODM^® (Micro-Optical Detection Method) that has potentially broad diagnostic application in laboratory systems and in rapid testing at the point of care. Inexpensive, single-use diagnostic devices for rapid testing have until now been largely limited to qualitative or semiquantitative tests that require visual interpretation. One unique application of MODM technology, the DRx^(TM) platform, resolves these issues and provides quantitative results at the point of care. It does so by incorporating microelectronics, optics, and dry reagent chemistry within a self-contained, integrated, single-use device. Up to four test results, which may be combinations of general chemistry tests and/or immunoassays (including ratioed results), can be displayed in numeric form on the device's liquid crystal display within 5 min after sample application. Having no switches or buttons, the device self-activates on addition of the sample. The overall dimensions of the device are 6.4 cm x 5.0 cm x 1.5 cm. An exploded view of the components of DRx is shown in Fig. 1 .

View larger version (55K): [in this window] [in a new window]   Figure 1. DRx single-use meter prototype design (exploded view). The key element shown in this figure is the molded optics subassembly, a single, molded transparent part consisting of 28 individual optical elements held in a precise spatial array that directs light from dual LED (light-emitting diode) sources to a total of four test zones on two dry reagent assay strips (not shown). It simultaneously samples light directly from the LEDs to a reference photodetector and gathers the light diffusively reflected from the test zones onto two sample photodetectors. It also supports and holds the dry reagent strips in precise alignment with its optical elements and with the sample pad. Between this part and the printed circuit board, an opaque molded part ("spider") provides optical isolation and shielding as well as protection for exposed bond wires attached to the bare die electronic components (LEDs, silicon photodiodes, microprocessor, and analog circuit components). Also shown in this figure are top and bottom case parts, aperture masks to control stray light and cross-talk between adjacent test zones, a donut-shaped desiccant, and "auto-start" leads (conductivity sensors that activate the device on addition of sample).

The first test developed for this new platform, DRx NTx, contains a dry reagent lateral flow immunoassay strip for measurement of NTx, plus a dry reagent general chemistry strip for measurement of creatinine in undiluted urine. NTx, the N-telopeptide cross-linking domain of type I collagen identified and characterized by Hanson et al. (1) , is a marker of bone collagen turnover. By virtue of the unique amino acid sequences forming the cross-linking domain, these particular peptides are derived only from mature bone collagen. Consequently, the ratio of NTx to creatinine in urine reflects the rate of bone turnover. This ratio also appears to be related to the risk of developing osteoporosis (2) and has been approved by the Food and Drug Administration for use in monitoring antiresorptive therapies. A more complete assessment of a patient's osteoporosis status requires, among other things, both bone mineral densitometry (a measure of current bone status) and measurement of a biochemical marker of bone turnover (a measure of the rate of change in bone status). Because NTx and other bone markers are measures of the rate of turnover (as opposed to extent), they can reflect changes in bone turnover that occur within a relatively short period of time, often as little as 4 weeks. Bone densitometry measurements, although quite precise, are a static measure. Often 1–2 years pass before marked bone loss can be detected by densitometry. This makes NTx useful in assessing response to therapeutic interventions (3)(4) such as estrogen replacement and bisphosphonates.

The reflectance of specific test zones on each strip is measured by a 4-channel reflectometer, and the clinical results are displayed as a ratio of NTx nmol BCE (bone collagen equivalent) per mmol creatinine (as calculated by an on-board microprocessor). The microprocessor also performs corrections for lot-specific reagent characteristics and several forms of optical variation, in addition to tests for proper electrical functioning and adequate sample volume.

In measurements of standard gray-scale materials (Munsell Color, GretagMacbeth), the miniaturized reflectometer had very good stability (<0.1% CV over 270 s, n = 73 for each channel), high reflectance reproducibility (0.5% CV, n = 10 for each channel), and excellent linearity (signal vs gray scale r² 0.999, slope = 1.00 ± 0.01, intercept <0.01 in units of reflectance). The range of NTx that can currently be measured extends from 30 to >1000 nmol/L BCE and from ~1 to 25 mmol/L for creatinine. Clinical precision (CV of the analyte concentration test result) for NTx and creatinine is in the range of 5–9% (NTx assay precision measured at 300 nmol/L; creatinine assay precision measured at 4 mmol/L; n = 20). The precision (CV) of ratioed results (NTx/creatinine) from 30 prototype DRx tests performed over a 3-month period with a buffered aqueous calibrator solution was 10.9% (38.3 ± 4.19 nmol BCE/mmol creatinine). Comparison of clinical sample test results (n = 20, performed in duplicate) to an NTx microtiter plate ELISA (Osteomark^(TM), Ostex International, Inc.) and to a Boehringer-Mannheim creatinine assay yielded correlation coefficient (r) values of 0.946 and 0.967, respectively. Correlation plot slopes and intercepts were (for NTx) 1.15 ± 0.06 and 40.4 ± 49.3 nmol/L BCE (S_yx = 169.4), and (for creatinine) 1.04 ± 0.04 and 1.28 ± 0.53 mmol/L (S_yx = 1.74), respectively. The correlation plot slope and intercept values for the ratio of NTx/creatinine were 0.998 ± 0.036 and 2.86 ± 3.36 (S_yx = 12.8), respectively, with an r value of 0.976.

We conclude that DRx NTx has performance that approaches that of clinical laboratory systems and can provide high-quality analytical information in a timely fashion at the point of care.

This work was supported by grant 1 R43 AR43721-01A2 from the National Institute of Arthritis and Musculoskeletal and Skin Diseases.

Footnotes

Metrika, Inc., 510 Oakmead Parkway, Sunnyvale, CA 94086

References

1. Hanson DA, Weis MAE, Bollen AM, Maslan SH, Singer FR, Eyre DR. A specific immunoassay for monitoring human bone resorption: quantitation of type I collagen cross-linked N-telopeptides in urine. J Bone Miner Res 1992;7:1251-1258. [ISI][Medline] [Order article via Infotrieve]
2. Garnero P, Sornay-Rendu E, Chapuy M-C, Delmas PD. Increased bone turnover in late postmenopausal women is a major determinant of osteoporosis. J Bone Miner Res 1996;11:337-349. [ISI][Medline] [Order article via Infotrieve]
3. Chesnut CH, Bell NH, Clark GS, Drinkwater BL, English SC, Johnston CC, et al. Hormone replacement therapy in postmenopausal women: urinary N-telopeptide of type I collagen monitors therapeutic effect and predicts response of bone mineral density. Am J Med 1997;102:29-37. [ISI][Medline] [Order article via Infotrieve]
4. Garnero P, Shih WJ, Gineyts E, Karpf DB, Delmas PD. Comparison of new biochemical markers of bone turnover in late postmenopausal osteoporotic women in response to alendronate treatment. J Clin Endocrinol Metab 1994;79:1693-1700. [Abstract]