Osteomark® NTx Serum EIA provides a quantitative measure of cross-linked Ntelopeptides of type I collagen (NTx) in serum as an indicator of human bone resorption. Serum NTx level is used to aid in predicting skeletal response (bone mineral density) to antiresorptive therapy and in monitoring bone resorption changes following initiation of antiresorptive therapy. Prior to initiating antiresorptive therapy, serum NTx level is used to determine the probability for a decrease in bone mineral density after one year in postmenopausal women treated with hormonal antiresorptive therapy relative to those treated with calcium supplement.

Device Description

The Osteomark NTx Serum EIA is a competitive-inhibition enzyme-linked immunosorbent assay (ELISA/EIA) for quantitative determination of NTx present in human serum. NTx is adsorbed to a 96-well microplate: Diluted samples are added to the microplate wells, followed by a horseradish peroxidase labeled monoclonal antibody. NTx in the specimen competes with the NTx epitope on the microtiter plate for antibody binding sites. Following a wash step, the amount of labeled antibody bound is measured by colorimetric generation of a peroxide substrate. NTx concentration is determined spectrophotometrically and calculated using a standard calibration curve. Assay values are reported in nanomoles Bone Collagen Equivalents per liter (nM BCE).

AI/ML Overview

The Osteomark NTx Serum EIA is a competitive-inhibition enzyme-linked immunosorbent assay (ELISA/EIA) designed for the quantitative determination of NTx in human serum. This assay is used to predict skeletal response to antiresorptive therapy and to monitor changes in bone resorption after therapy initiation. It also helps determine the probability of bone mineral density decrease in postmenopausal women on hormonal antiresorptive therapy versus those on calcium supplements.

1. Table of Acceptance Criteria and Reported Device Performance

Performance Metric	Acceptance Criteria	Reported Device Performance
Intra-assay variability	Not explicitly stated as "acceptance criteria," but the study established this metric.	2.6% (measured by testing four human serum specimens distributed throughout the calibration range)
Inter-assay variability	Not explicitly stated as "acceptance criteria," but the study established this metric.	6.9% (measured by testing eight human serum specimens distributed throughout the calibration range)
Total assay precision (Level I Serum Control)	Not explicitly stated as "acceptance criteria," but the study established this metric.	13.99% Coefficient of Variation
Total assay precision (Level II Serum Control)	Not explicitly stated as "acceptance criteria," but the study established this metric.	11.92% Coefficient of Variation
Antigen Recovery	Not explicitly stated as "acceptance criteria," but the study established this metric.	100% average recovery (across the assay range)
Dilutional Linearity	Not explicitly stated as "acceptance criteria," but the study established this metric.	98% average linearity
Prediction of BMD decrease (high baseline NTx, no HRT)	Not explicitly stated as "acceptance criteria," but implicitly shown as a significant risk factor.	A high baseline NTx (≥18.1 nM BCE) indicated a 6 times higher risk of BMD loss if not treated with HRT.
Monitoring response to HRT	Not explicitly stated as "acceptance criteria," but implicitly shown as a significant decrease in NTx levels.	Mean values fell significantly after 3 months of HRT (from 15.9 nM BCE to 12.2 nM BCE, a -22.9% decrease).
Monitoring response to Bisphosphonate	Not explicitly stated as "acceptance criteria," but implicitly shown as a significant decrease in NTx levels.	Mean values after 6 months of alendronate treatment (11.0 nM BCE) were significantly lower than baseline (16.1 nM BCE).
Correlation with greater BMD gain	Not explicitly stated as "acceptance criteria," but implicitly shown as a significant correlation.	Subjects with highest baseline NTx (≥16.6 nM BCE) had the greatest gain in PA spine BMD (5.76%) with alendronate.

2. Sample Size Used for the Test Set and Data Provenance

The document describes several studies:

Reference Range Studies:
- Premenopausal Women: N = 257 (multi-center, cross-sectional study at five regional sites). The country of origin is not specified but implied to be the US given the FDA submission. This was a prospective study to determine reference ranges.
- Men: N = 176 (multi-center, cross-sectional study at three regional sites). The country of origin is not specified but implied to be the US. This was a prospective study to determine reference ranges.
Intra-subject Variability Studies:
- Postmenopausal Women: n=271 (short-term), n=261 (long-term). Provenance not specified. Retrospective/Prospective not specified for this specific study, but the overall context suggests prospective data collection in clinical settings.
- Men: n=32 (short-term), n=27 (long-term). Provenance not specified. Retrospective/Prospective not specified for this specific study.
Assay Reproducibility and Precision Studies:
- Intra-assay variability: Four human serum specimens. Provenance not specified. Not directly tied to a clinical population, but rather assay samples.
- Inter-assay variability: Eight human serum specimens. Provenance not specified. Not directly tied to a clinical population, but rather assay samples.
- Total assay precision: Level I Serum Control and Level II Serum Control tested at four clinical laboratories. Provenance not specified. These are control materials, not patient samples.
Antigen Recovery: Nine serum specimens. Provenance not specified. These are assay samples.
Dilutional Linearity: Five serum specimens. Provenance not specified. These are assay samples.
Clinical Studies (HRT):
- Postmenopausal Women treated with HRT: The study enrolled a cohort of postmenopausal women. The baseline NTx mean was 15.9 nM BCE. Post-treatment mean was 12.2 nM BCE. While a specific N for the HRT group is not provided in this summary, the context suggests a clinical trial. The study results (Reference 4) indicate the source is likely from U.S. clinical trials. This was a prospective clinical trial.
Clinical Studies (Bisphosphonate):
- Postmenopausal Women treated with Bisphosphonate: The study was conducted at a regional specialty hospital in the northeast United States. The study was randomized and double-blind. N is not explicitly stated in the summary, but it involved women randomized to either placebo or alendronate. This was a prospective clinical trial.

3. Number of Experts Used to Establish Ground Truth for the Test Set and Qualifications of Those Experts

The document does not describe the use of human "experts" to establish ground truth for the device performance studies in the traditional sense of a diagnostic interpretation.

For the clinical efficacy studies (HRT and Bisphosphonate), the "ground truth" was clinical outcomes:

Bone Mineral Density (BMD) changes: Measured by objective methods (e.g., DEXA scans) over time. This is an objective clinical measurement, not expert consensus on an image or test result.
Response to therapy: Defined by changes in NTx levels and subsequent changes in BMD.

4. Adjudication Method for the Test Set

Not applicable. The reported studies evaluate the analytical performance of the assay and its correlation with clinical outcomes (BMD changes), not the interpretation of results by human readers requiring adjudication.

5. Multi-Reader Multi-Case (MRMC) Comparative Effectiveness Study

No. The Osteomark NTx Serum EIA is an in-vitro diagnostic assay for quantitative measurement. It does not involve human readers interpreting images or data where AI assistance would be relevant for a MRMC study.

6. Standalone (Algorithm Only Without Human-in-the-Loop Performance) Study

Yes, implicitly. The performance characteristics (variability, recovery, linearity) and the clinical utility of the assay itself (e.g., "Osteomark NTx Serum EIA mean baseline values... fell significantly after 3 months of therapy") are reported as standalone performance of the diagnostic kit. There is no human-in-the-loop component for the assay's execution or the interpretation of the numerical NTx values for these studies. The physician uses the derived NTx value in clinical management.

7. Type of Ground Truth Used

Analytical Performance: The ground truth for analytical performance (variability, recovery, linearity) is based on established laboratory analytical methods and expected theoretical values for spiked samples or diluted controls.
Clinical Studies: The ground truth for the clinical studies (HRT and Bisphosphonate) is outcomes data and objective clinical measurements:
- Bone Mineral Density (BMD) assessed after one year.
- Changes in NTx levels post-therapy. This is an outcome of the intervention, not an externally established ground truth for the device itself.

8. Sample Size for the Training Set

The document describes the device performance validation and clinical studies. There is no description of a "training set" in the context of machine learning (AI) for this diagnostic assay. The terms "training set" and "test set" are not applicable in the typical AI sense to this type of in-vitro diagnostic device. Instead, the studies cited relate to establishing reference ranges and validating analytical and clinical performance.

9. How the Ground Truth for the Training Set Was Established

Not applicable, as no "training set" in the AI sense is described. The analytical and clinical performance data were established through direct laboratory measurements and clinical trial outcomes, respectively, as outlined above.

Summary

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1983457

Summary of Sales of the Summary of Safety and Effectiveness Summary of Safety and Effectiveness

Assay Principles

The Osteomark NTx Serum EIA is a competitive-inhibition enzyme-linked immunosorbent assay (ELISA/EIA) for quantitative determination of NTx present in human serum.

NTx is adsorbed to a 96-well microplate: Diluted samples are added to the microplate wells, followed by a horseradish peroxidase labeled monoclonal antibody. NTx in the specimen competes with the NTx epitope on the microtiter plate for antibody binding sites. Following a wash step, the amount of labeled antibody bound is measured by colorimetric generation of a peroxide substrate. NTx concentration is determined spectrophotometrically and calculated using a standard calibration curve. Assay values are reported in nanomoles Bone Collagen Equivalents per liter (nM BCE).

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Kit Components

Supplied Materials sufficient for 96 wells

Instructions for Use	1 booklet
Antigen Coated 96-Well Plate, 12 1x8 well strips	1 plate
Specimen Diluent	40 mL bottle
Calibrators:
0 nM BCE Calibrator	20.0 mL vial
1 nM BCE Calibrator	0.4 mL vial
5 nM BCE Calibrator	0.4 mL vial
10 nM BCE Calibrator	0.4 mL vial
20 nM BCE Calibrator	0.4 mL vial
40 nM BCE Calibrator	0.4 mL vial
Level I Serum Control	0.4 mL vial
Level II Serum Control	0.4 mL vial
Antibody Conjugate Concentrate	0.4 mL vial
Antibody Conjugate Diluent	25 mL bottle
30X Wash Concentrate	125 mL bottle
Chromogen Reagent	0.9 mL vial
Buffered Substrate	30 mL bottle
Stopping Reagent	30 mL bottle
Plate Sealers	1 pad

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Expected Values

A multi-center, cross-sectional study was conducted at five regional sites to determine the reference range for normal premenopausal women (mean age 36 years, range 25-49). Male reference range was determined from a multi-center, cross-sectional study conducted at three regional sites (mean age 51 years, range 31-80).6

	Mean*	Std Dev	Range(mean ± 2 Std Dev)	N
Women	12.6	3.2	6.2 - 19.0	257
Men	14.8	4.7	5.4 - 24.2	176

*expressed as nM BCE

When the premenopausal women expected value range is log-transformed, the range is 7.7 - 19.3 nM BCE. The log-transformed male reference range is 8.1 - 24.8 nM BCE. These reference ranges are provided as guidelines only. Each laboratory should establish their own reference range.

In a study of postmenopausal women', short-term (3 days) and long-term (2 months) intra-subject variability was determined to be 7.3% (n=271) and 8.7% (n=261), respectively. In a subset of the above male reference range study population, the shortterm (4 days) intra-subject variability was 9.1% (n=32), and the long-term (3 months) intra-subject variability was 9.5% (n=27).

Performance Characteristics

Assay Reproducibility and Precision

Intra-assay variability was determined by testing four human serum specimens following NCCLS Precision Performance Guideline EP5-T2 with BCE values distributed throughout the calibration range of the assay. From these test results the Osteomark NTx Serum EIA intra-assay variability is established as 2.6%.

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Inter-assay variability was determined by testing eight human serum specimens with BCE values distributed throughout the calibration range of the assay. From these test results the Osteomark NTx Serum EIA inter-assay variability is established as 6.9%.

Total assay precision was evaluated by testing the Level I Serum Control and the Level II Serum Control at four clinical laboratories. Estimate of the total precision percent coefficient of variation for the Level I Serum Control was 13.99% and for the Level II Serum Control was 11.92%.

Antigen Recovery was evaluated by adding known amounts of NTx to each of nine serum specimens of known NTx concentration. Recovery represented the observed assay value of the "spiked" specimens, calculated as a percent of the expected serum value (baseline serum value plus added antigen (NTx) value). Results demonstrated an average antigen recovery of 100% across the assay range.

Dilutional linearity was evaluated by performing serial dilutions of five serum specimens with high nM BCE values into a serum specimen with a known low nM BCE value. Percent linearity was determined as the measured value divided by the expected value multiplied by 100. Results demonstrated an average linearity of 98%.

Clinical Studies

Use of Osteomark NTx Serum EIA in Postmenopausal Women Treated With HRT A clinical trial was conducted to determine the ability of the Osteomark NTx Serum EIA to monitor the effect of hormone replacement therapy (HRT) on bone resorption and to determine the probability for a decrease in bone mineral density (BMD) after one year if treated with only calcium supplements relative to those treated with supplements and HRT.4 Results of the study supported these clinical uses. Prior to HRT initiation,

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Osteomark NTx Serum EIA mean baseline values in this group were 15.9 nM BCE, significantly higher than the premenopausal mean of 12.6 nM BCE. In the HRT group, mean values fell significantly after 3 months of therapy to 12.2 nM BCE, a -22.9% decrease. Mean values in the calcium group remained constant throughout the 12 month study; 15.4 nM BCE at baseline and 15.8 nM BCE after 12 months. Examination of the contrast between the HRT and calcium only groups provides information regarding risk of BMD loss. In the lowest NTx quartile (≤12.5 nM BCE), there was no statistically significant difference between the HRT and calcium groups in the likelihood of bone loss over 1 year. A high baseline NTx (≥18.1 nM BCE) indicated a 6 times higher risk of BMD loss if not treated with HRT.

Use of Osteomark NTx Serum EIA in Postmenopausal Women Treated With Bisphosphonate

A study was conducted at a regional specialty hospital in the northeast United States to determine if early changes in Osteomark NTx Serum EIA following treatment with the bisphosphonate alendronate sodium predicts an increase in BMD.5 In this randomized, double-blind clinical study, women were randomized to either placebo or 5-10 mg alendronate sodium. In the alendronate treated group, mean Osteomark NTx Serum EIA values after 6 months of treatment, 11.0 nM BCE, were significantly lower than baseline, 16.1 nM BCE. Stratification of baseline Osteomark NTx Serum EIA demonstrate that subjects with the highest values (≥ 16.6 nM BCE) had the greatest gain in PA spine BMD (5.76%).

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References

1. Garnero P., et. al. Comparison of new biochemical markers of bone turnover in late postmenopausal osteoporotic women in response to alendronate treatment. J Clin Endo and Met. 79:1963-1700. 1994.
1. Garnero P., et. al. Increased bone turnover in late postmenopausal women is a major determinant of osteoporosis. J Bone Miner Res. 11:337-349. 1996.
1. Prestwood K.M., et. al. The short-term effects of conjugated estrogen on bone turnover in older women. J. Clin Endo and Met. 79:366-371. 1994
1. Chesnut C.H., 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. 102:29-37. 1997.
1. Greenspan S.L., et. al. Early changes in biochemical markers of bone turnover predict the long-term response to alendronate therapy in representative elderly women: A randomized clinical trial. J Bone Miner Res. 13:1431-1438. 1998.
1. Orwoll E.S., et. al. Collagen N-telopeptide excretion in men: The effects of age and intra-subject variability. J. Clin Endo and Met. In Press.
1. Weiss S., et. al. Determination of the intrasubject variability in NTx excretion in postmenopausal women. J Bone Miner Res. Vol. 12. Suppl. 1, pp. S506. 1997.

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OSTEOMARK NTx SERUM EIA QUICK REFERENCE GUIDE

1. Thoroughly read the Assay Procedure before you begin.
1. Allow all specimens and kit components to come to room temperature. Mix all reagents thoroughly.
1. Prepare working strength wash solution. Dilute 30X Wash Concentrate 1:30 with deionized water.
1. Plan the plate configuration, and create a plate map.
1. Prepare working strength antibody conjugate solution at a 1:101 dilution. You will need approximately 1 mL per strip.
1. Prepare 1:5 dilutions of all Calibrators, Controls and specimens in Specimen Diluent using microtubes or equivalent.
1. Pipette, in duplicate wells, 100 µL of each diluted Calibrator, Control or specimen into the microplate according to the plate map.
1. Pipette 100 uL of working strength antibody conjugate solution into each microwell. Gently swirl to mix, cover the plate with a plate sealer and incubate the plate at room temperature for 90 ± 5 minutes.
1. Prepare Chromogen Reagent/Buffered Substrate solution at a 1:101 dilution during the last 5 minutes of incubation. You will need approximately 2 mL per strip.
1. Wash microwells five (5) times with working strength wash solution. Blot on absorbent paper after the final wash.
1. Add 200 uL diluted Chromogen Reagent/Buffered Substrate to each microwell, gently swirl to mix, and incubate at room temperature for 30 ± 2 minutes.
1. Add 100 uL of Stopping Reagent to each microwell. Gently swirl the plate to mix.
1. Incubate at room temperature for five minutes and read the absorbance of each microwell at 450 nm. Calculate the results using a 4-parameter logistic curve fit.

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Public Health Service

2 1999 FEB

Food and Drug Administration 2098 Gaither Road Rockville MD 20850

Ms. Nancy Mallinak Vice President, Regulatory And Clinical Affairs Ostex International 2203 Airport Way South Suite 400 Seattle, Washington 98134

Re: K983457

Trade Name: Osteomark NTx Serum EIA Regulatory Class: I Product Code: JMM Dated: December 18, 1998 Received: December 22, 1998

Dear Ms. Mallinak:

We have reviewed your Section 510(k) notification of intent to market the device referenced above and we have determined the device is substantially equivalent (for the indications for use stated in the enclosure) to legally marketed predicate devices marketed in interstate commerce prior to May 28, 1976, the enactment date of the Medical Device Amendments, or to devices that have been reclassified in accordance with the provisions of the Federal Food, Drug, and Cosmetic Act (Act). You may, therefore, market the device, subject to the general controls provisions of the Act. The general controls provisions of the Act include requirements for annual registration, listing of devices, good manufacturing practice, labeling, and prohibitions against misbranding and adulteration.

If your device is classified (see above) into either class II (Special Controls) or class III (Premarket Approval), it may be subject to such additional controls. Existing major regulations affecting your device can be found in the Code of Federal Regulations, Title 21, Parts 800 to 895. A substantially equivalent determination assumes compliance with the Current Good Manufacturing Practice requirements, as set forth in the Quality System Regulation (QS) for Medical Devices: General regulation (21 CFR Part 820) and that, through periodic OS inspections, the Food and Drug Administration (FDA) will verify such assumptions. Failure to comply with the GMP regulation may result in regulatory action. In addition, FDA may publish further announcements concerning your device in the Federal Register. Please note: this response to your premarket notification submission does not affect any obligation you might have under sections 531 through 542 of the Act for devices under the Electronic Product Radiation Control provisions, or other Federal laws or regulations.

Image /page/7/Picture/10 description: The image is a black and white logo for the U.S. Department of Health & Human Services. The logo features a stylized design of three human profiles facing to the right, stacked on top of each other. The profiles are simple and abstract, with only the outlines of the faces visible. The text "DEPARTMENT OF HEALTH & HUMAN SERVICES - USA" is arranged in a circular pattern around the profiles.

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Page 2

Under the Clinical Laboratory Improvement Amendments of 1988 (CLIA-88), this device may require a CLIA complexity categorization. To determine if it does, you should contact the Centers for Disease Control and Prevention (CDC) at (770) 488-7655.

This letter will allow you to begin marketing your device as described in your 510(k) premarket notification. The FDA finding of substantial equivalence of your device to a legally marketed predicate device results in a classification for your device and thus, permits your device to proceed to the market.

If you desire specific advice for your device on our labeling regulation (21 CFR Part 801 and additionally 809.10 for in vitro diagnostic devices), please contact the Office of Compliance at (301) 594-4588. Additionally, for questions on the promotion and advertising of your device, please contact the Office of Compliance at (301) 594-4639. Also, please note the regulation entitled, "Misbranding by reference to premarket notification"(21 CFR 807.97). Other general information on your responsibilities under the Act may be obtained from the Division of Small Manufacturers Assistance at its toll-free number (800) 638-2041 or (301) 443-6597, or at its internet address "http://www.fda.gov/cdrh/dsma/dsmamain.html".

Sincerely yours,

Steven Butman

Steven I. Gutman, M.D., M.B.A. Director Division of Clinical Laboratory Devices Office of Device Evaluation Center for Devices and Radiological Health

Enclosure

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Page 1 of 1

K983457 S10 (k) NUMBER (IF KNOWN) : OSTEOMARK® NTx SERUM EIA DEVICE NAME: INDICATIONS FOR USE:

Sean Cooper
(Division Sign-Off)

Division of Clinical Laboratory Devices 510(k) Number

(PLEASE DO NOT WRITE BELOW THIS LINE-CONTINUE ON ANOTHER PAGE IF NEEDED.)

Concurrence of CDRH, - Office of Device Evaluation (ODE)

Prescription Use (Per 21 CFR 801.109) OR

Over-The-Counter-Use (Optional Format 1-2-96)

Regulation Number and Section

§ 862.1400 Hydroxyproline test system.

(a)
Identification. A hydroxyproline test system is a device intended to measure the amino acid hydroxyproline in urine. Hydroxyproline measurements are used in the diagnosis and treatment of various collagen (connective tissue) diseases, bone disease such as Paget's disease, and endocrine disorders such as hyperparathyroidism and hyperthyroidism.(b)
Classification. Class I (general controls). The device is exempt from the premarket notification procedures in subpart E of part 807 of this chapter subject to § 862.9.