IDS-iSYS Ostase BAP

K200475 · Immunodiagnostic Systems , Ltd. · CIN · Sep 30, 2020 · Clinical Chemistry

Device Facts

• Record ID: K200475
• Device Name: IDS-iSYS Ostase BAP
• Applicant: Immunodiagnostic Systems , Ltd.
• Product Code: CIN · Clinical Chemistry
• Decision Date: Sep 30, 2020
• Decision: SESE
• Submission Type: Traditional
• Regulation: 21 CFR 862.1050
• Device Class: Class 2

Intended Use

The IDS-iSYS Ostase® BAP assay is an in vitro diagnostic device intended for the quantitative determination of bone-specific alkaline phosphatase (BAP), an indicator of osteoblastic activity, in human serum on the IDS system. Results are to be used in conjunction with other clinical and laboratory data to aid the clinician in the management of postmenopausal osteoporosis and Paget’s disease.

Device Story

IDS-iSYS Ostase® BAP is an in vitro diagnostic assay for quantitative measurement of bone-specific alkaline phosphatase (BAP) in human serum. Device utilizes an automated IDS system to process samples; reagent cartridge contains streptavidin-coated magnetic particles, biotinylated monoclonal anti-BAP antibody, and p-nitrophenyl phosphate substrate. Assay principle involves binding of BAP to magnetic particles and biotinylated antibody, followed by enzymatic detection. Output is a BAP concentration value (µg/L). Used in clinical laboratories by trained technicians. Results assist clinicians in managing postmenopausal osteoporosis and Paget's disease by providing an indicator of osteoblastic activity. Benefits include automated processing compared to manual predicate methods, improving laboratory efficiency and standardization.

Clinical Evidence

Bench testing only. Analytical performance validated per CLSI guidelines (EP05-A3, EP06-A, EP17-A2, EP07-A2). Precision studies (n=240 replicates per sample) showed within-lab CVs 3.0-7.2%. Linearity confirmed 3-70 µg/L (y=0.98x-0.9, R²=1.00). Interference testing confirmed no significant impact from common substances (e.g., biotin up to 350 ng/mL, RF up to 1200 IU/mL). Method comparison against predicate (n=150) yielded Passing-Bablok slope 0.99, intercept 0.17, r=0.99. Reference intervals established in 419 healthy donors.

Technological Characteristics

Quantitative enzyme immunoassay; reagent cartridge includes streptavidin-coated magnetic particles, biotinylated monoclonal anti-BAP antibody, and p-nitrophenyl phosphate substrate. Automated kinetic absorbance measurement at 405 nm. Traceable to predicate via in-house secondary standards. Complies with CLSI standards for precision (EP05-A3), linearity (EP06-A), detection limits (EP17-A2), and reference intervals (EP28-A3c).

Indications for Use

Indicated for the quantitative determination of bone-specific alkaline phosphatase (BAP) in human serum to aid clinicians in the management of postmenopausal osteoporosis and Paget's disease in adult patients.

Regulatory Classification

Identification

An alkaline phosphatase or isoenzymes test system is a device intended to measure alkaline phosphatase or its isoenzymes (a group of enzymes with similar biological activity) in serum or plasma. Measurements of alkaline phosphatase or its isoenzymes are used in the diagnosis and treatment of liver, bone, parathyroid, and intestinal diseases.

Predicate Devices

• Tandem-MP Ostase Immunoenzymetric Assay (k972666)

Submission Summary (Full Text)

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September 30, 2020

Immunodiagnostic Systems Ltd. Mick Henderson RA Manager 10 Didcot Way, Boldon Business Park Boldon, Tyne and Wear NE35 9PD United Kingdom

Re: K200475

Trade/Device Name: IDS-iSYS Ostase® BAP Regulation Number: 21 CFR 862.1050 Regulation Name: Alkaline phosphatase or isoenzymes test system Regulatory Class: Class II Product Code: CIN Dated: August 27, 2020 Received: August 31, 2020

Dear Mick Henderson:

We have reviewed your Section 510(k) premarket notification of intent to market the device referenced above and 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) that do not require approval of a premarket approval application (PMA). You may, therefore, market the device, subject to the general controls provisions of the Act. Although this letter refers to your product as a device, please be aware that some cleared products may instead be combination products. The 510(k) Premarket Notification Database located at https://www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfpmn/pmn.cfm identifies combination product submissions. 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. Please note: CDRH does not evaluate information related to contract liability warranties. We remind you, however, that device labeling must be truthful and not misleading.

If your device is classified (see above) into either class II (Special Controls) or class III (PMA), it may be subject to additional controls. Existing major regulations affecting your device can be found in the Code of Federal Regulations, Title 21, Parts 800 to 898. In addition, FDA may publish further announcements concerning your device in the Federal Register.

Please be advised that FDA's issuance of a substantial equivalence determination does not mean that FDA has made a determination that your device complies with other requirements of the Act or any Federal

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542 of the Act); 21 CFR 1000-1050.

statutes and regulations administered by other Federal agencies. You must comply with all the Act's requirements, including, but not limited to: registration and listing (21 CFR Part 807); labeling (21 CFR Part 801 and Part 809); medical device reporting of medical device-related adverse events) (21 CFR 803) for devices or postmarketing safety reporting (21 CFR 4, Subpart B) for combination products (see https://www.fda.gov/combination-products/guidance-regulatory-information/postmarketing-safety-reportingcombination-products); good manufacturing practice requirements as set forth in the quality systems (QS) regulation (21 CFR Part 820) for devices or current good manufacturing practices (21 CFR 4, Subpart A) for combination products; and, if applicable, the electronic product radiation control provisions (Sections 531-

Also, please note the regulation entitled, "Misbranding by reference to premarket notification" (21 CFR Part 807.97). For questions regarding the reporting of adverse events under the MDR regulation (21 CFR Part 803), please go to https://www.fda.gov/medical-device-safety/medical-device-reportingmdr-how-report-medical-device-problems.

For comprehensive regulatory information about medical devices and radiation-emitting products, including information about labeling regulations, please see Device Advice (https://www.fda.gov/medicaldevices/device-advice-comprehensive-regulatory-assistance) and CDRH Learn (https://www.fda.gov/training-and-continuing-education/cdrh-learn). Additionally, you may contact the Division of Industry and Consumer Education (DICE) to ask a question about a specific regulatory topic. See the DICE website (https://www.fda.gov/medical-device-advice-comprehensive-regulatoryassistance/contact-us-division-industry-and-consumer-education-dice) for more information or contact DICE by email ([e-mail]) or phone [phone] or 301-796-7100).

Sincerely,

Marianela Perez- Torres, Ph.D. Acting Deputy Director Division of Chemistry and Toxicology Devices OHT7: Office of In Vitro Diagnostics and Radiological Health Office of Product Evaluation and Quality Center for Devices and Radiological Health

Enclosure

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## Indications for Use

510(k) Number (if known) k200475

Device Name IDS-iSYS Ostase® BAP

#### Indications for Use (Describe)

The IDS-iSYS Ostase® BAP assay is an in vitro diagnostic device intended for the quantitative determination of bonespecific alkaline phosphatase (BAP), an indicator of osteoblastic activity, in human serum on the IDS system. Results are to be used in conjunction with other clinical and laboratory data to aid the clinician in the management of postmenopausal osteoporosis and Paget's disease.

| Type of Use (Select one or both, as applicable) | |
|-------------------------------------------------|---------------------------------------------|
| ☑ Prescription Use (Part 21 CFR 801 Subpart D) | Over-The-Counter Use (21 CFR 801 Subpart C) |

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Image /page/3/Picture/0 description: The image shows the logo for Immunodiagnostic Systems (IDS). The logo features the letters "ids" in a stylized, connected font, with a red circle above the "i". Below the letters, the words "immunodiagnosticsystems" are written in a smaller, red font. A registered trademark symbol is located to the right of the logo.

# 510(k) SUMMARY

| 510k Number | k200475 |
|--------------|----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
| Introduction | According to the requirements of 21CFR807.92, the following<br>information provides sufficient detail to understand the basis for a<br>determination of substantial equivalence. |
| Submitter | Immunodiagnostic Systems Limited<br>10 Didcot Way<br>Boldon Business Park<br>Boldon<br>Tyne and Wear<br>NE35 9PD<br>United Kingdom |
| | Contact Person: Mick Henderson<br>Phone: [phone]<br>Fax: [phone]<br>Email: [e-mail] |
| | Secondary Contact: Alexandra Bennett<br>Phone: [phone]<br>Fax: [phone]<br>Email: [e-mail]<br>Date prepared: 28 September 2020 |
| Device Name | Proprietary names: IDS-iSYS Ostase® BAP |
| | Common names: As above |
| | Classification: 21CFR862.1050 Alkaline phosphatase or<br>isoenzymes test system. |
| | Class II |
| | Product Code: CIN |

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Image /page/4/Picture/0 description: The image shows the logo for Immunodiagnostic Systems (IDS). The logo features the letters 'ids' in a stylized, interconnected design, with a small red circle above the 'i'. Below the main logo, the words 'immunodiagnosticsystems' are written in a smaller, sans-serif font, with the first word in red and the second in gray. The 'R' in a circle indicates that the logo is a registered trademark.

- Predicate Device The IDS-iSYS Ostase® BAP is substantially equivalent to other products in commercial distribution intended for similar use. We claim equivalency to the currently marketed Tandem-MP Ostase Immunoenzymetric Assay (k972666), commercially known as Ostase® BAP EIA.
Device Description The IDS-iSYS Ostase® BAP assay consists of one reagent cartridge and one set of calibrators (CAL A & CAL B).

The reagent cartridge contains multiple reagents:

- MPM1 (Magnetic particles coated with streptavidin in a phosphate buffer with sodium azide as preservative);
- Ab-BIOT Monoclonal anti-BAP labelled with biotin, in buffer containing horse serum with bovine and mouse proteins and sodium azide as a preservative (<0.1 %)
- -SUBS (p-nitrophenyl phosphate in a stabilising buffer containing preservatives).

Calibrators A and B are buffered bovine protein matrix containing human BAP with sodium azide as preservative (<0.1 %).

- Indications for Use The IDS-iSYS Ostase® BAP assay is an in vitro diagnostic device intended for the quantitative determination of bone-specific alkaline phosphatase (BAP), an indicator of osteoblastic activity, in human serum on the IDS system. Results are to be used in conjunction with other clinical and laboratory data to aid the clinician in the management of postmenopausal osteoporosis and Paget's disease.
- Conditions for use: For in vitro diagnostic use only. Rx Only

### Special instrument Requirements:

IDS System (k091849)

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Image /page/5/Picture/0 description: The image shows the logo for Immunodiagnostic Systems (IDS). The logo features the letters 'ids' in a stylized, gray font, with a red circle above the 'i'. Below the letters, the words 'immunodiagnosticsystems' are written in a smaller, red font. A circled 'R' trademark symbol is located in the upper right corner of the logo.

# Comparison Tables

# Similarities compared to the chosen (FDA cleared; marketed) predicate device (k972666)

Assay

| Assay<br>Performance | Predicate Device<br>Tandem-MP Ostase<br>Immunoenzymetric Assay<br>(k972666), commercially<br>known as Ostase® BAP EIA | Candidate Device<br>IDS-iSYS Ostase® BAP<br>(k200475) |
|----------------------------------------------------------------------------------------------------------------------------|-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
| Intended Use | For quantitative determination of<br>Bone Alkaline Phosphatase<br>concentration | For quantitative determination of<br>Bone Alkaline Phosphatase<br>concentration |
| Analyte | Bone Alkaline Phosphatase | Bone Alkaline Phosphatase |
| Sample<br>Volume | 50 μL | 50 μL |
| Sample Type | Human Serum | Human Serum |
| Specificity,<br>Interfering<br>substances &<br>Cross<br>Reactivity | Interfering Substances<br>Acetaminophen 20 mg/dL<br>Alendronate 5 mg/dL<br>Asprin 50 mg/dL<br>Bilirubin –<br>unconjugated 40 mg/dL<br>Calcitonin – salmon 112 IU/dL<br>Calcium 20 mg/dL<br>Ibuprofen 40 mg/dL<br>Pamidronate 18 mg/dL<br>Progesterone 25 mg/dL | Interfering Substances<br>Acetaminophen Same<br>Alendronate Same<br>Asprin Same<br>Bilirubin –<br>unconjugated Same<br>Calcitonin - salmon Same<br>Calcium Same<br>Ibuprofen Same<br>Pamidronate Same<br>Progesterone Same |
| Performance | Predicate Device<br>Tandem-MP Ostase<br>Immunoenzymetric Assay<br>(k972666), commercially known<br>as Ostase® BAP EIA | Candidate Device<br>IDS-iSYS Ostase® BAP<br>(k200475) |
| Indications<br>for Use | The Ostase® BAP EIA Assay is an<br><i>in vitro</i> device indicated for the<br>quantitative measurement of bone-<br>specific alkaline phosphatase<br>(BAP), an indicator of osteoblastic<br>activity, in human serum. This<br>device is intended to be used as an<br>aid in the management of<br>postmenopausal osteoporosis and<br>Paget's disease. | The IDS-iSYS Ostase® BAP<br>assay is an <i>in vitro</i> diagnostic<br>device intended for the<br>quantitative determination of<br>bone-specific alkaline phosphatase<br>(BAP), an indicator of osteoblastic<br>activity, in human serum on the<br>IDS system. Results are to be used<br>in conjunction with other clinical<br>and laboratory data to aid the<br>clinician in the management of<br>postmenopausal osteoporosis and<br>Paget's disease |
| Method of<br>detection<br>(Test<br>methodology) | Manual | Automated |
| Kit reagent<br>components | Conjugate (1 x 14 mL)<br>anti-BAP(mouse monoclonal IgG)<br>with biotin in a bovine/horse protein<br>matrix with 0.09% sodium azide<br><br>Microplates (1 x 96 wells)<br>Streptavidin coated plastic well<br>strips<br><br>Zero Calibrator (1 x 14 mL)<br>A bovine protein matrix containing<br>no detectable concentration of BAP<br>and 0.09% sodium azide<br><br>Calibrators (1-5) (5 x 1 mL)<br>A bovine protein matrix containing<br>approximately 7, 15, 30, 60 and 90<br>ug human BAP/L and 0.09%<br>sodium azide | Reagent Cartridge<br><br>MPM1 (1 x 2.6 mL)<br>Magnetic particles coated with<br>streptavidin in a Phosphate buffer<br>with sodium azide as preservative<br>(<0.09%)<br><br>Ab-BIOT (1 x 10.5 mL)<br>Monoclonal anti-BAP labelled<br>with biotin, in buffer containing<br>horse serum with bovine and<br>mouse proteins and sodium azide<br>as a preservative (<0.09%) |
| Low Control (1 x 1 mL)<br>A bovine protein matrix containing<br>approximately 11 ug human BAP/L<br>and 0.09% sodium azide | SUBS (1 x 40 mL)<br>p-nitrophenyl phosphate in a<br>stabilizing buffer containing<br>preservatives | |
| High Control (1 x 1 mL)<br>A bovine protein matrix containing<br>approximately 45 ug human BAP/L<br>and 0.09% sodium azide | Calibrators (1 each of 2<br>concentrations levels – 2.5ml per<br>bottle).<br>A buffered bovine protein matrix<br>containing human BAP with<br>sodium azide as preservative<br>(<0.09%) | |
| Wash Concentrate (1 x 50 mL)<br>Phosphate buffered saline<br>containing Tween | | |
| Substrate (1 x 20 mL)<br>p-nitrophenyl phosphate in a<br>stabilizing buffer containing<br>preservatives | | |
| Quench Reagent (1 x 14ml)<br>1 N Sodium hydroxide | | |
| Range of assay | 0.7 – 90 μg/L | 3 – 70 μg/L |
| Sensitivity | LoB N/A<br>LoD 0.7 µg/L<br>LoQ N/A | LoB 0.3 µg/L<br>LoD 0.4 µg/L<br>LoQ 0.5 µg/L |
| Expected<br>values | Males<br>Mean 12.3 µg/L<br>Median 11.6 µg/L<br>95th percentile 20.1 µg/L<br>Pre-Menopausal<br>Mean 8.7 µg/L<br>Median 8.5 µg/L<br>95th percentile 14.3 µg/L<br>Post-Menopausal<br>Mean 13.2 µg/L<br>Median 12.5 µg/L<br>95th percentile 22.4 µg/L | Males<br>Mean 13.7 µg/L<br>Median 13 µg/L<br>Range 7.9 to 23.5 μg/L<br>Pre-Menopausal<br>Mean 11.5 µg/L<br>Median 11.1 µg/L<br>Range 5.9 to 20.5 μg/L<br>Post-Menopausal<br>Mean 15.7 µg/L<br>Median 14.3 µg/L<br>Range 7.9 to 34.2 μg/L |

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Image /page/6/Picture/0 description: The image shows the logo for Immunodiagnostic Systems (IDS). The logo features the letters "ids" in a stylized, gray font, with a red circle above the "i". Below the letters, the words "immunodiagnosticsystems" are written in a smaller, red font. A registered trademark symbol is located to the right of the "s" in "ids".

# Differences compared to the chosen (FDA cleared; marketed) predicate device (k972666)

Assay

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Image /page/7/Picture/0 description: The image shows the logo for Immunodiagnostic Systems (IDS). The logo features the letters "ids" in a stylized, gray font, with a red circle above the "i". Below the letters, the words "immunodiagnostic systems" are written in a smaller, red font. The logo is simple and modern, with a focus on the company's name and area of expertise.

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Image /page/8/Picture/0 description: The image shows the logo for Immunodiagnostic Systems (IDS). The logo features the letters "ids" in a stylized, sans-serif font, with a red circle above the "i". Below the letters, the words "immunodiagnostic systems" are written in a smaller, sans-serif font.

| Precision | Within Run Precision n =20<br>2.6% to 6.5% in the concentration<br>range 7.4 to 79.5 μg/L<br><br>Between Run Precision n = 20<br>2% to 6.4% in the concentration<br>range 8.4 to 81.1 µg/L | Repeatability n =80<br>1.7% to 2.8% in the concentration<br>range 6.2 to 59.8 µg/L<br><br>Within Laboratory n = 80<br>3.0% to 7.6% in the concentration<br>range 6.2 to 59.8 µg/L | | |
|-------------------------------------------------------------------------|--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|--|
| Specificity,<br>Interfering<br>substances<br>And<br>Cross<br>Reactivity | Interfering Substances | Interfering Substances | | |
| | Bilirubin –<br>conjugated<br>Biotin<br>Cholesterol<br>Estradiol<br>Etidronate<br>Haemoglobin<br>HAMA<br>PTH 1-34<br>PTH 1-84<br>Raloxifene<br>Red Blood Cells<br>Rheumatoid Factor<br>(Rf)<br>Risedronate<br>Total Protein<br>Triglycerides<br>25-hydroxyvitamin D | Bilirubin –<br>20 mg/dL<br>conjugated<br>No Claim<br>Biotin<br>No Claim<br>Cholesterol<br>400 ng/dL<br>Estradiol<br>105 mg/dL<br>Etidronate<br>500 mg/dL<br>Haemoglobin<br>No Claim<br>HAMA<br>No Claim<br>PTH 1-34<br>No Claim<br>PTH 1-84<br>No Claim<br>Raloxifene<br>No Claim<br>Red Blood Cells<br>No Claim<br>Rheumatoid Factor<br>(Rf)<br>No Claim<br>Risedronate<br>No Claim<br>Total Protein<br>14 g/dL<br>Triglycerides<br>2000 mg/dL<br>25-hydroxyvitamin D<br>80,500 IU/dL | Bilirubin –<br>40 mg/dL<br>conjugated<br>Biotin<br>400 ng/mL<br>Cholesterol<br>340 mg/dL<br>Estradiol<br>400 µg/mL<br>Etidronate<br>90 mg/dL<br>Haemoglobin<br>300 mg/dL<br>HAMA<br>4000 ng/mL<br>PTH 1-34<br>20 µg/dL<br>PTH 1-84<br>11.8 µg/dL<br>Raloxifene<br>20 µg/mL<br>Red Blood Cells<br>0.3%<br>Rheumatoid Factor<br>(Rf)<br>1200 IU/mL<br>Risedronate<br>50 µg/dL<br>Total Protein<br>12 g/dL<br>Triglycerides<br>667 mg/dL<br>25-hydroxyvitamin D<br>125 ng/mL | |
| | Cross Reactivity<br>Intestinal ALP<br>100U/L yields a result of 1.0 µg/L<br>Liver ALP<br>100 U/L yields a result of 6.2µg/L<br>Placental ALP<br>Non detectable | Cross Reactivity<br>Intestinal ALP<br>Spiked 745 µg/L yields 0.1%<br>Liver ALP<br>Spiked 90 U/L yields 0.5%<br>Placental ALP<br>Non detectable | | |
| Method<br>comparison | Against Tandem-R Ostase<br>(k961573)<br>n = 136<br>$Ostase® BAP EIA =1.02 x (Tandem-R Ostase) +0.28 µg/L$<br>Correlation coefficient (r) = 0.97 | | Against Ostase® BAP EIA:<br>n = 150<br>$IDS-iSYS Ostase® BAP =0.99 x (Ostase® BAP EIA) +0.17µg/L$<br>Correlation coefficient (r) = 0.99 | |

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Image /page/9/Picture/0 description: The image shows the logo for Immunodiagnostic Systems (IDS). The logo features the letters "ids" in a stylized, connected font, with a red dot above the "i". Below the letters, the words "immunodiagnosticsystems" are written in a smaller, red font. The logo is simple and modern, with a focus on the company's name and area of expertise.

| Linearity | No Claim | Observed = |
|-----------|----------|---------------------------------|
| | | 0.98 x (Expected) -0.9 µg/L |
| | | Regression coefficient R2: 1.00 |

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Image /page/10/Picture/0 description: The image shows the logo for Immunodiagnostic Systems (IDS). The logo features the letters "ids" in a stylized, connected font, with a red circle above the "i". Below the letters, the words "immunodiagnosticsystems" are written in a smaller, sans-serif font. A registered trademark symbol is located to the right of the "s" in "ids".

### Performance Characteristics (if/when applicable):

- 1. Analytical performance:
#### a. Precision/Reproducibility:

Precision was evaluated in accordance with a modified protocol based on CLSI EP-5A3, "Evaluation of Precision Performance of Quantitative Measurement Methods". Ten (10) native serum samples were assayed using three reagent lots in duplicate, twice per day for 20 days (n= 80 replicates per sample) on three systems.

| | | Mean | Repeatability | | Within Laboratory | |
|-----------|----|-----------------|---------------|------|-------------------|------|
| Sample | N | Conc.<br>(µg/L) | SD (µg/L) | %CV | SD (µg/L) | %CV |
| Sample 1 | 80 | 6.1 | 0.2 | 2.5% | 0.2 | 2.8% |
| Sample 2 | 80 | 8.4 | 0.1 | 1.6% | 0.2 | 2.8% |
| Sample 3 | 80 | 11.4 | 0.2 | 2.0% | 0.4 | 3.9% |
| Sample 4 | 80 | 12.1 | 0.3 | 2.2% | 0.5 | 4.5% |
| Sample 5 | 80 | 18.0 | 0.4 | 2.4% | 0.8 | 4.5% |
| Sample 6 | 80 | 19.8 | 0.5 | 2.5% | 0.9 | 4.6% |
| Sample 7 | 80 | 45.1 | 1.2 | 2.7% | 1.6 | 3.5% |
| Sample 8 | 80 | 52.4 | 1.0 | 1.9% | 1.9 | 3.5% |
| Sample 9 | 80 | 54.3 | 0.9 | 1.7% | 1.6 | 2.9% |
| Sample 10 | 80 | 58.6 | 1.1 | 1.8% | 1.5 | 2.5% |

Results from one representative reagent lot (Lot #3):

Results for the combined 3 lots:

| Sample | N | Mean Conc. (µg/L) | Repeatability SD (µg/L) | %CV | Within Laboratory SD (µg/L) | %CV |
|-----------|-----|-------------------|-------------------------|------|-----------------------------|------|
| Sample 1 | 240 | 6.2 | 0.2 | 2.8% | 0.4 | 5.9% |
| Sample 2 | 240 | 8.7 | 0.1 | 1.7% | 0.5 | 6.2% |
| Sample 3 | 240 | 11.7 | 0.3 | 2.5% | 0.8 | 6.6% |
| Sample 4 | 240 | 12.5 | 0.3 | 2.4% | 0.9 | 7.1% |
| Sample 5 | 240 | 18.4 | 0.4 | 2.4% | 1.3 | 6.8% |
| Sample 6 | 240 | 20.6 | 0.5 | 2.5% | 1.5 | 7.2% |
| Sample 7 | 240 | 45.5 | 1.0 | 2.3% | 1.4 | 3.0% |
| Sample 8 | 240 | 53.2 | 1.1 | 2.1% | 1.8 | 3.4% |
| Sample 9 | 240 | 54.7 | 0.9 | 1.7% | 2.1 | 3.9% |
| Sample 10 | 240 | 59.8 | 1.1 | 1.9% | 2.3 | 3.8% |

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Image /page/11/Picture/0 description: The image shows the logo for Immunodiagnostic Systems (IDS). The logo features the letters "ids" in a stylized, sans-serif font, with the "i" having a red circular dot above it. Below the letters, the words "immunodiagnosticsystems" are written in a smaller, red font. The logo has a registered trademark symbol in the upper right corner.

#### b. Linearity/assay reportable range:

A linearity study was conducted based on CLSI EP6-A for the candidate device. A high human serum sample and a low human serum sample were used to create 11 evenly spaced dilutions by mixing the high and low sample to cover the assay measuring range as indicated below:

| Sample | Dilution | Dilution Factor (%) |
|--------|---------------|---------------------|
| 1: | Low (L) | 0 |
| 2: | 0.90L + 0.10H | 10 |
| 3: | 0.80L + 0.20H | 20 |
| 4: | 0.70L + 0.30H | 30 |
| 5: | 0.60L + 0.40H | 40 |
| 6: | 0.50L + 0.50H | 50 |
| 7: | 0.40L + 0.60H | 60 |
| 8: | 0.30L + 0.70H | 70 |
| 9: | 0.20L + 0.80H | 80 |
| 10: | 0.10L + 0.90H | 90 |
| 11: | High (H) | 100 |

Results:

Observed = 0.98 x (Expected) -0.9 ng/mL Regression coefficient R2: 1.00

The IDS-iSYS Ostase® BAP assay linear range was determined as 0.9 to 78.5 µg/L, with a measuring (reportable) range of 3 to 70 µg/L.

### c. Traceability, Stability, Expected values (controls, calibrators, or methods):

#### Calibrator traceability and value assignment

The IDS-iSYS Ostase® BAP kit calibrators are value assigned against in house secondary standard (IRs) using an internal OC procedure. The IR's are value assigned against the predicate device (Ostase® BAP EIA assay) using the predicate assay procedure. Therefore, the kit calibrators are traceable to the predicate device via the secondary standards.

#### Calibrator Value Assignment and Verification Method

For kit calibrator value assignment, the kit calibrators are tested as unknowns in a minimum of 20 assay runs on one IDS-iSYS system. The secondary standards (IRs) are assayed in each of the runs, and the values of the Kit Calibrators are assigned with direct reference to the secondary standards (IRs). The assigned kit calibrator values are then verified following the IDS QC procedure; by running the assay on three different IDS systems and analyzing IQCs of known values across the range of the assay.

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### Stability

Full kit stability was performed in which the kit calibrators were tested in combination with all kit combination reagents.

The stability based on real time studies determined a shelf life of 12 months.

### d. Detection limit:

The limit of blank (LoB). limit of detection (LoD) and limit of quantitation (LoO) were determined with guidance from CLSI EP17-A, "Protocols for Determination of Limits of Detection and Limits of Quantitation" in three (3) kit lots.

Each LoB sample was measured in duplicate, for a total of five assays in a five-day period, generating a total of 60 replicates per kit lot. Each kit lot was tested on a different instrument. Analyse-It software was used to calculate the LoB.

The LoD was determined using ten (10) samples with very low BAP concentrations. Each LoD sample was measured in duplicate, for a total of five assays in a five-day period per kit lot. Each kit lot was tested on a different instrument. Analyse-It software was used to calculate the LoD.

The LoQ was calculated using ten (10) samples with low BAP concentration. Each LoQ sample was measured in duplicate, for a total of five assays in a five-day period. Each kit lot was tested on a different instrument. Analyse-It software was used to calculate the predicted LoQ. The claim limit for each kit lot is the actual closest value to the Analyse-It's precited value.

The limit of blank (LoB), limit of detection (LoD) and limit of quantitation (LoQ) were determined with guidance from CLSI EP17-A, "Protocols for Determination of Limits of Detection and Limits of Quantitation" using 60 replicates of blank and 10 low level samples.

| Sensitivity | BAP Concentration (µg/L) |
|-----------------------------|--------------------------|
| LoB (Limit of Blank) | 0.3 |
| LoD (Limit of Detection) | 0.4 |
| LoQ (Limit of Quantitation) | 0.5 |

### e. Analytical specificity:

Interference and cross-reactivity studies were performed in accordance with the CLSI EP07-A3 Interference.

To determine potential interference, two serum samples at two different concentrations of BAP were spiked with the potential interferent. Control samples (blank) were spiked with a volume of Phosphate Buffer saline (PBS) (0 ng/mL) or relevant diluent equal to that of the spiked interferent. The mean of 26 replicate assays, for both spiked and control samples,

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were then compared. The differences observed between the mean spiked and control sample values were examined and assessed according to acceptance criteria.

For Rheumatoid factor (Rf) or total cholesterol, the interference was tested by recovery of BAP from a high serum pool spiked into a serum sample with known Rf levels or cholesterol levels. % Interference was calculated using below formula:

% Interference = (mean spiked concentration - mean un-spiked concentration) x 100 mean un-spiked concentration

% Recovery was calculated using the formula below:

Recovery value = Observed mean spiked value - Observed mean unspiked value

% Recovery = (Recovery value / Expected Recovery value (Analyte added)) x 100

The following compounds were tested and found not to interfere significantly with the test, based on the predefined acceptance criteria of non-significant interference of <10% bias between the test and control samples:

| Potential Interfering Substance | Highest concentration tested that demonstrated no significant interference |
|---------------------------------|----------------------------------------------------------------------------|
| Acetaminophen | 20 mg/dL |
| Alendronate | 5 mg/dL |
| Bilirubin (Conjugated) | 40 mg/dL |
| Bilirubin (Unconjugated) | 40 mg/dL |
| Biotin | 400 ng/mL |
| Calcium Chloride | 20 mg/dL |
| Cholesterol | 325 mg/dL |
| Estradiol | 400 µg/mL |
| Etidronate | 90 mg/dL |
| Haemoglobin | 300 mg/dL |
| HAMA | 4000 ng/mL |
| Ibuprofen | 40 mg/dL |
| Pamidronate | 18 mg/dL |
| Progesterone | 25 mg/dL |
| PTH 1-34 | 20 µg/dL |
| PTH 1-84 | 11.8 µg/dL |
| Raloxifene | 20 µg/mL |
| Red Blood Cells | 0.3% |
| Rheumatoid Factor (RF) | 1200 IU/mL |
| Risedronate | 50 µg/mL |
| Salicylic Acid (Asprin) | 50 mg/dL |
| Salmon Calcitonin | 112 IU/dL |
| Total Protein | 12 g/dL |
| Triglycerides | 667 mg/dL |
| 25-hydroxyvitamin D | 125 ng/mL |

Cross-reactivity testing was performed on Liver, Placental and Intestinal derived ALP.

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the indicated substances were spiked into serum samples and measured with the IDS-iSY S Ostase® BAP, following the CLSI EP7-A2. The percent cross-reactivity was calculated using below formula:

% cross reactivity =

(Mean concentration of spiked sample - mean concentration of un-spiked sample) x100% Spike concentration

The substances with structures similar to bone alkaline phosphatase (BAP) were spiked into serum samples and measured with the IDS Ostase® BAP, following the CLSI EP7-A2. The exogenous substances were tested at the concentration listed below and determined to have the following percent cross-reactivity:

| Cross-Reactant | Spiked Concentration | % Cross Reactivity |
|----------------|----------------------|--------------------|
| Liver ALP | 745 µg/L | 0.1% |
| Placental ALP | 90 U/L | 0.5% |
| Intestinal ALP | 500 µg/L | Undetectable |

f. Assay cut-off: Not applicable

### 2. Comparison studies:

The IDS-iSYS Ostase® BAP assay was compared against the IDS Ostase® BAP EIA assay, following CLSI EP-9A2 "Method Comparison and Bias Estimation Using Patient A total of 150 samples, selected to represent a wide range of BAP Samples". concentrations [3.0 to 67.6 ug/L], was assayed by each method. Passing-Bablok regression analysis was performed on the comparative data:

| N | Slope | 95% CI | Intercept (µg/L) | 95% CI | Corr. Coefficient<br>(r) |
|-----|-------|--------------|------------------|-------------|--------------------------|
| 150 | 0.99 | 0.97 to 1.02 | 0.17 | -0.1 to 0.5 | 0.99 |

### 3. Expected values/Reference range:

The BAP concentration was measured in serum samples collected from 419 apparently healthy donors from the United States using the IDS Ostase® BAP assay. The study population included 140 males (35 to 75 years of age), 140 pre-menopausal women (35 to 45 years of age) and 139 post-menopausal women (55 to 75 years of age). The observed ranges (2.5th to 97.5th percentile) were established, according to CLSI guideline C28-A3c, "How to Define and Determine Reference Intervals in the Clinical Laboratory" are summarized in the below table:

| Population | N | Age (years) | BAP Concentration (μg/L) |
|------------|---|-------------|--------------------------|
|------------|---|-------------|--------------------------|

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| | | Median | Min.-Max | Mean | Median | SD | Observed Range |
|-----------------|-----|--------|----------|------|--------|-----|----------------|
| Males | 140 | 49 | 35 to 75 | 13.7 | 13.0 | 4.1 | 7.9 to 23.5 |
| Pre-menopausal | 140 | 39 | 35 to 45 | 11.5 | 11.1 | 3.9 | 5.9 to 20.5 |
| Post-menopausal | 139 | 58 | 55 to 75 | 15.7 | 14.3 | 6.7 | 7.9 to 34.2 |

The above ranges should be considered as guidelines only; it is recommended that each laboratory establish its own expected range based upon its own patient population.

## Conclusion:

The IDS-iSYS Ostase® BAP assay data presented and provided are complete and supports the basis for substantial equivalence to the predicate device.