Tina-quant Hemoglobin A1cDx Gen.3

K193053 · Roche Diagnostics Operations (Rdo) · PDJ · Mar 26, 2020 · Clinical Chemistry

Device Facts

• Record ID: K193053
• Device Name: Tina-quant Hemoglobin A1cDx Gen.3
• Applicant: Roche Diagnostics Operations (Rdo)
• Product Code: PDJ · Clinical Chemistry
• Decision Date: Mar 26, 2020
• Decision: SESE
• Submission Type: Traditional
• Regulation: 21 CFR 862.1373
• Device Class: Class 2

Intended Use

The Tina-quant Hemoglobin A1cDx Gen.3 assay is intended for use as an aid in diagnosis of diabetes and as an aid in identifying patients who may be at risk for developing diabetes. It is an in vitro diagnostics reagent system intended for quantitative determination of mmol/mol hemoglobin A1c (IFCC) and % hemoglobin A1c (DCCT/NGSP) in hemolysate or venous whole blood on the cobas c 503 clinical chemistry analyzer. HbA1c determinations are useful for monitoring of long-term blood glucose control in individuals with diabetes mellitus.

Device Story

Tina-quant Hemoglobin A1cDx Gen.3 is an in vitro diagnostic reagent system for quantitative HbA1c measurement on the cobas c 503 clinical chemistry analyzer. It utilizes a turbidimetric inhibition immunoassay (TINIA). The system processes anticoagulated venous whole blood or manually hemolyzed samples. For whole blood, hemolysis occurs onboard using a reagent containing tetradecyltrimethylammonium bromide (TTAB), which lyses erythrocytes but not leukocytes. The assay measures total hemoglobin (Hb) bichromatically and HbA1c via antibody-polyhapten complex formation. The analyzer calculates the HbA1c/Hb ratio, reporting results in IFCC (mmol/mol) or DCCT/NGSP (%) units. Used in clinical laboratories by trained personnel. Results assist clinicians in diagnosing diabetes, assessing risk, and monitoring long-term glycemic control. The assay is compatible with common hemoglobin variants (HbS, HbC, HbD, HbE) and is unaffected by common endogenous substances or drugs.

Clinical Evidence

Bench testing only. Precision studies (CLSI EP05-A3) demonstrated reproducibility with CVs generally <2%. Linearity was confirmed from 4.2% to 20.1% HbA1c. Method comparison against the NGSP reference method (Tosoh HPLC) showed strong correlation (Passing-Bablok regression). Interference studies confirmed no significant impact from common endogenous substances or drugs. No clinical trials were required.

Technological Characteristics

Turbidimetric inhibition immunoassay (TINIA). Reagents: R1 (anti-HbA1c antibody, ovine), R3 (polyhapten), and A1CD (TTAB-based hemolyzing reagent). Measurement: Bichromatic photometry. Platform: cobas c 503 analyzer. Calibration: RCM4 for HbA1c, linear for Hb. Traceability: NGSP/IFCC. Measuring range: 23-196 mmol/mol (IFCC) and 4.2-20.1% (DCCT/NGSP).

Indications for Use

Indicated for use as an aid in the diagnosis of diabetes and identifying patients at risk for developing diabetes. Used for monitoring long-term blood glucose control in individuals with diabetes mellitus. Applicable to venous whole blood or hemolysate samples.

Regulatory Classification

Identification

A hemoglobin A1c test system is a device used to measure the percentage concentration of hemoglobin A1c in blood. Measurement of hemoglobin A1c is used as an aid in the diagnosis of diabetes mellitus and as an aid in the identification of patients at risk for developing diabetes mellitus.

Special Controls

(b) Classification. Class II (special controls). Hemoglobin A1c test systems must comply with the following special controls: 1) The device must have initial and annual standardization verification by a certifying glycohemoglobin standardization organization deemed acceptable by FDA. 2) The premarket notification submission must include performance testing to evaluate precision, accuracy, linearity and interference, including the following: i) Performance testing of device precision must, at a minimum, use blood samples with concentrations near 5.0%, 6.5%, 8.0% and 12% hemoglobin A1c. This testing must evaluate precision over a minimum of 20 days using at least 3 lots of the device and 3 instruments, as applicable. ii) Performance testing of device accuracy must include a minimum of 120 blood samples that span the measuring interval of the new device and compare results of the new device to results of the standardized test method. Results must demonstrate little or no bias versus the standardized method. iii) Total error of the new device must be evaluated using single measurements by the new device compared to results of the standardized test method, and this evaluation must demonstrate a total error less than or equal to 6%. iv) Performance testing must demonstrate that there is little to no interference from common hemoglobin variants, including Hemoglobin C, Hemoglobin D, Hemoglobin E, Hemoglobin A2 and Hemoglobin S. 3) When assay interference from Hemoglobin F or interference with other hemoglobin variants with low frequency in the population is observed, a warning statement must be placed in a black box and must appear in all labeling material for these devices describing the interference and any affected populations.

*Classification.* Class II (special controls). The special controls for this device are:(1) The device must have initial and annual standardization verification by a certifying glycohemoglobin standardization organization deemed acceptable by FDA. (2) The premarket notification submission must include performance testing to evaluate precision, accuracy, linearity, and interference, including the following: (i) Performance testing of device precision must, at a minimum, use blood samples with concentrations near 5.0 percent, 6.5 percent, 8.0 percent, and 12 percent hemoglobin A1c. This testing must evaluate precision over a minimum of 20 days using at least three lots of the device and three instruments, as applicable. (ii) Performance testing of device accuracy must include a minimum of 120 blood samples that span the measuring interval of the device and compare results of the new device to results of a standardized test method. Results must demonstrate little or no bias versus the standardized method. (iii) Total error of the new device must be evaluated using single measurements by the new device compared to results of the standardized test method, and this evaluation must demonstrate a total error less than or equal to 6 percent. (iv) Performance testing must demonstrate that there is little to no interference from common hemoglobin variants, including Hemoglobin C, Hemoglobin D, Hemoglobin E, Hemoglobin A2, and Hemoglobin S. (3) When assay interference from Hemoglobin F or interference with other hemoglobin variants with low frequency in the population is observed, a warning statement must be placed in a black box and must appear in all labeling material for these devices describing the interference and any affected populations.

Predicate Devices

• COBAS INTEGRA 800 Tina-quant Hemoglobin A1cDx Gen.2 assay (k121291)

Reference Devices

• Tosoh Automated Glycohemoglobin Analyzer HLC-723G8

Submission Summary (Full Text)

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Image /page/0/Picture/0 description: The image contains the logo of the U.S. Food and Drug Administration (FDA). On the left is the Department of Health & Human Services logo. To the right of that is the FDA logo, which is a blue square with the letters "FDA" in white. To the right of the blue square is the text "U.S. FOOD & DRUG ADMINISTRATION" in blue.

March 26, 2020

Roche Diagnostics Operations Inc. Leslie Patterson Regulatory Affairs Principal 9115 Hague Rd Indianapolis, Indiana 46250

### Re: K193053

Trade/Device Name: Tina-quant Hemoglobin A1cDx Gen.3 Regulation Number: 21 CFR 862.1373 Regulation Name: Hemoglobin A1c Test System Regulatory Class: Class II Product Code: PDJ, LCP Dated: February 13, 2020 Received: February 14, 2020

Dear Leslie Patterson:

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 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

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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 531-542 of the Act); 21 CFR 1000-1050.

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) k193053

Device Name Tina-quant Hemoglobin A1cDx Gen.3

### Indications for Use (Describe)

The Tina-quant Hemoglobin A1cDx Gen.3 assay is intended for use as an aid in diagnosis of diabetes and as an aid in identifying patients who may be at risk for developing diabetes. It is an in vitro diagnostics reagent system intended for quantitative determination of mmol/mol hemoglobin A1c (IFCC) and % hemoglobin A1c (DCCT/NGSP) in hemolysate or venous whole blood on the cobas c 503 clinical chemistry analyzer. HbA1c determinations are useful for monitoring of long-term blood glucose control in individuals with diabetes mellitus.

| Type of Use (Select one or both, as applicable) | |
|----------------------------------------------------------------------------------------------------------------------|---------------------------------------------------------------------------------------------------------------------|
| <div> <span> <span style="font-size: 16px;">☑</span> Prescription Use (Part 21 CFR 801 Subpart D) </span> </div> | <div> <span> <span style="font-size: 16px;">☐</span> Over-The-Counter Use (21 CFR 801 Subpart C) </span> </div> |

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# Tina-quant Hemoglobin A1cDx Gen.3 510(k) Summary (k193053)

This summary of 510(k) safety and effectiveness information is being submitted in accordance with the requirements of 21 CFR 807.92.

In accordance with 21 CFR 807.87, Roche Diagnostics hereby submits official notification as required by Section 510(k) of the Federal Food, Drug and Cosmetics Act of our intention to market the device described in this Premarket Notification 510(k).

The purpose of this Traditional 510(k) Premarket Notification is to obtain FDA review and clearance for the Tina-quant Hemoglobin A1cDx Gen.3 assay.

| Submitter Name | Roche Diagnostics Operations Inc. |
|--------------------------------------|-------------------------------------------------------------------------------------------------------|
| Address | 9115 Hague Road<br>P.O. Box 50416<br>Indianapolis, IN 46250-0457 |
| Contact | Leslie Patterson<br>Phone: [phone]<br>FAX: [phone]<br>Email: [e-mail] |
| Date Prepared | February 13, 2020 |
| Proprietary Name | Tina-quant Hemoglobin A1cDx Gen.3 |
| Common Name | Glycosylated Hemoglobin Assay |
| Classification Name | Hemoglobin A1c test system |
| Product Codes,<br>Regulation Numbers | PDJ, 862.1373<br>LCP, 864.7470 |
| Predicate Device | COBAS INTEGRA 800 Tina-quant Hemoglobin A1cDx Gen.2 assay, k121291 |
| Establishment Registration | 1823260, Roche Diagnostics Corporation |

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#### 1. DEVICE DESCRIPTION

Tina-quant Hemoglobin A1cDx Gen.3 assay is an in vitro diagnostics reagent system intended for quantitative determination of mmol/mol hemoglobin A1c (IFCC) and % hemoglobin A1c (DCCT/NGSP) in hemolysate or whole blood on the cobas c 503 clinical chemistry analyzer.

The assay offers separate applications that are specific to the sample types whole blood and hemolysate. The Whole Blood Application differs from the Hemolysate Application in the hemolyzing step. For the Whole Blood Application, whole blood samples are placed on the analyzer and hemolysis occurs onboard the analyzer. For the Hemolysate Application, hemolyzed samples are placed on the analyzer and hemolysis occurs manually before placing the samples onboard the analyzer. The two applications yield the same results. Hemolyzing reagent is part of the test system and is either placed on board the analyzer for the Whole Blood Application or used manually for the Hemolysate Application.

Anticoagulated whole blood is hemolyzed either manually or automatically prior to determination of HbAlc by a turbidimetric inhibition immunoassay. Liberated hemoglobin (Hb) in the hemolyzed sample is converted to a derivative having a characteristic absorption spectrum and measured bichromatically. The instrument calculates the % HbAlc from the HbAlc/Hb ratio according to a user selected protocol, either IFCC or NGSP protocols.

#### Test Principle 1.1.

This method uses tetradecyltrimethylammonium bromide (TTAB) as the detergent in the hemolyzing reagent to eliminate interference from leukocytes (TTAB does not lyse leukocytes). Sample pretreatment to remove labile HbA1c is not necessary. All hemoglobin variants which are glycated at the ß -chain N-terminus and which have antibody-recognizable regions identical to that of HbA1c are determined by this assay. Consequently, the metabolic state of patients having uremia or the most frequent hemoglobinopathies (HbAC, HbAC, HbAD) can be determined using this assay.

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#### 1.2. Hemoglobin A1c

The HbA1c determination is based on the turbidimetric inhibition immunoassay (TINIA) for hemolyzed whole blood.

- . Sample and addition of R1 (buffer/antibody):
Glycohemoglobin (HbA1c) in the sample reacts with anti-HbA1c antibody to form soluble antigen-antibody complexes. Since the specific HbA1c antibody site is present only once on the HbA1c molecule, formation of insoluble complexes does not take place.

- Addition of R3 (buffer/polyhapten) and start of reaction: .
The polyhaptens react with excess anti-HbA1c antibodies to form an insoluble antibodypolyhapten complex which can be determined turbidimetrically.

#### 1.3. Hemoglobin

Liberated hemoglobin in the hemolyzed sample is converted to a derivative having a characteristic absorption spectrum which is measured bichromatically during the preincubation phase (sample + R1) of the above immunological reaction. A separate Hb reagent is consequently, not necessary.

#### Final HbA1c Result 1.4.

The final result is expressed as mmol/mol HbA1c and is calculated from the HbA1c/Hb ratio as follows:

Protocol 1 (mmol/mol HbA1c acc. to IFCC): HbA1c (mmol/mol) = (HbA1c/Hb) × 1000

Protocol 2 (% HbA1c acc. to DCCT/NGSP): HbA1c (%) = (HbA1c/Hb) × 91.5 + 2.15

#### 1.5. Standardization

Traceability: This method has been standardized against the approved IFCC reference method for the measurement of HbA1c in human blood and can be transferred to results traceable to DCCT/NGSP by calculation.

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#### 2. INDICATIONS FOR USE

The Tina-quant Hemoglobin A1cDx Gen.3 assay is intended for use as an aid in diagnosis of diabetes and as an aid in identifying patients who may be at risk for developing diabetes. It is an in vitro diagnostics reagent system intended for quantitative determination of mmol/mol hemoglobin A1c (IFCC) and % hemoglobin A1c (DCCT/NGSP) in hemolysate or venous whole blood on the cobas c 503 clinical chemistry analyzer. HbA1c determinations are useful for monitoring of long-term blood glucose control in individuals with diabetes mellitus.

#### TECHNOLOGICAL CHARACTERISTICS 3.

The following table compares the Tina-quant Hemoglobin A1cDx Gen.3 assay with its predicate device, COBAS INTEGRA 800 Tina-quant Hemoglobin A1cDx Gen.2 (k121291).

| Feature | Predicate Device:<br>COBAS INTEGRA 800 Tina-quant<br>Hemoglobin A1cDx Gen.2 k121291 | Submitted Device:<br>Tina-quant Hemoglobin A1cDx Gen.3 |
|-----------------------------|----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
| Intended Use | This test is to be used as an aid in diagnosis of<br>diabetes and as an aid in identifying patients<br>who may be at risk for developing diabetes. It<br>is an in vitro diagnostic reagent system<br>intended for quantitative determination of<br>mmol/mol hemoglobin A1c (IFCC) and %<br>hemoglobin A1c (DCCT/NGSP) in hemolysate<br>or venous whole blood. HbA1c determinations<br>are useful for monitoring of long-term blood<br>glucose control in individuals with diabetes<br>mellitus. | Same |
| Sample Types | Anticoagulated venous blood<br>Acceptable anticoagulants for both the<br>hemolysate and whole blood applications<br>include:<br>• Li-Heparin<br>• K2-EDTA<br>• K3-EDTA<br>• Fluoride/potassium oxalate<br>• Na-Heparin<br>• NaF/Na2-EDTA | Anticoagulated venous blood<br>Acceptable anticoagulants for both the<br>hemolysate and whole blood applications<br>include:<br>• Li-Heparin<br>• K2-EDTA<br>• K3-EDTA<br>• Fluoride/potassium oxalate<br>• Na-Heparin<br>• EDTA Fluoride |
| Instrument<br>Platform | COBAS Integra 800<br>• Absorbance Photometry | cobas c 503 |
| Calibrator | Cfas HbA1c | Same |
| Feature | Predicate Device:<br>COBAS INTEGRA 800 Tina-quant<br>Hemoglobin A1cDx Gen.2 k121291 | Submitted Device:<br>Tina-quant Hemoglobin A1cDx Gen.3 |
| Calibration<br>Frequency | • After 29 days on-board the analyzer<br>• After reagent lot change<br>• As required following quality control<br>procedures | Same |
| Calibration Mode | Logit/Log 5 | Hb: linear<br>HbA1c : RCM4 |
| Controls | PreciControl HbA1c norm<br>PreciControl HbA1c path | Same |
| Reagent Stability | Unopened:<br>• 2-8 °C until expiration date<br>On-board in use:<br>• 2-8 °C for 28 days | Same |
| Reporting Units | • % HbA1c (NGSP/DCCT)<br>• mmol/mol (IFCC) | Same |
| Antibody | Polyclonal anti-HbA1c from sheep blood | Same |
| Test Principle | The anticoagulated whole blood specimen is<br>hemolyzed automatically on the COBAS<br>INTEGRA 800 analyzers with COBAS<br>INTEGRA Hemolyzing Reagent Gen.2. This<br>method uses TTAB as the detergent in the<br>hemolyzing reagent to eliminate interference<br>from leukocytes (TTAB does not lyse<br>leukocytes). Sample pretreatment to remove<br>labile HbA1c is not necessary. All hemoglobin<br>variants which are glycated at the β-chain N-<br>terminus and which have antibody-recognizable<br>regions identical to that of HbA1c are<br>determined by this assay. Consequently, the<br>metabolic state of diabetic patients having<br>uremia or the most frequent<br>hemoglobinopathies (HbAS, HbAC, HbAE,<br>HbAD) can be determined by this assay. | This method uses<br>tetradecyltrimethylammonium bromide (TTAB)<br>as the detergent in the hemolyzing reagent to<br>eliminate interference from leukocytes (TTAB<br>does not lyse leukocytes). Sample<br>pretreatment to remove labile HbA1c is not<br>necessary. All hemoglobin variants which are<br>glycated at the β -chain N-terminus and which<br>have antibody-recognizable regions identical to<br>that of HbA1c are determined by this assay.<br>Consequently, the metabolic state of patients<br>having uremia or the most frequent<br>hemoglobinopathies (HbAS, HbAC, HbAE,<br>HbAD) can be determined using this assay. |
| Determination of<br>HbA1c | Turbidimetric immunoinhibition (TINIA).<br>Antigen-antibody complexes are formed<br>and excess Ab aggregate with polyhapten to<br>form insoluble complexes | HbA1c determination is based on the<br>turbidimetric inhibition immunoassay (TINIA)<br>for hemolyzed whole blood. Glycohemoglobin<br>in the sample reacts with anti-HbA1c antibody<br>to form soluble antigen-antibody complexes.<br>Polyhaptens react with excess anti-HbA1c<br>antibodies to form an insoluble antibody-<br>polyhapten complex which can be measured<br>turbidimetrically. |
| Determination of<br>Hb | Bichromatic photometric determination<br>after conversion to a colored derivate | Liberated hemoglobin in the hemolyzed<br>sample is converted to a derivative having a<br>characteristic absorption spectrum which is<br>measured bichromatically. |
| Feature | Predicate Device:<br>COBAS INTEGRA 800 Tina-quant<br>Hemoglobin A1cDx Gen.2 k121291 | Submitted Device:<br>Tina-quant Hemoglobin A1cDx Gen.3 |
| Determination<br>of % HbA1c | The final result is expressed as % HbA1c and<br>is calculated from the HbA1c/Hb ratio per<br>DCCT/NGSP as follows:<br>$HbA1c (%) = (HbA1c/Hb) × 91.5 + 2.15$ | Same |
| Measuring Range | Hemoglobin: 4-35 g/dL<br>HbA1c: 0.3-3.4 g/dL<br>This corresponds to a measuring range of<br>23-258 mmol/mol HbA1c (IFCC)<br>and 4.3-24.8 % HbA1c (DCCT/NGSP) at a<br>typical hemoglobin concentration of 13.2 g/dL. | Hemoglobin: 4-40 g/dL (2.48-24.8 mmol/L)<br>HbA1c: 0.3-2.6 g/dL (0.186-1.61 mmol/L)<br>This corresponds to a measuring range of 23-<br>196 mmol/molHbA1c (IFCC) and 4.2-20.1 %<br>HbA1c (DCCT/NGSP) at a typical hemoglobin<br>concentration of 13.2 g/dL (8.2 mmol/L). |
| Traceability | The assigned HbA1c and total hemoglobin<br>values of the cobas c Tina-quant Hemoglobin<br>A1cDx Gen.3 assay is certified with the<br>National Glycohemoglobin Standardization<br>Program (NGSP). NGSP certification is<br>repeated annually. | Same |
| Reagent<br>Composition | R1 Antibody Reagent:<br>• MES (2-morpholinoethane sulfonic acid)<br>buffer: 0.025 mol/L<br>• TRIS (Tris(hydroxymethyl) aminomethane)<br>buffer: 0.015 mol/L, pH 6.2<br>• HbA1c antibody (ovine serum): ≥ 0.5 mg/ml<br>• detergents; stabilizers; preservatives<br>SR Polyhapten Reagent:<br>• MES buffer: 0.025 mol/L<br>• TRIS buffer: 0.015 mol/L, pH 6.2<br>• HbA1c polyhapten: > 8µg/mL<br>• stabilizers; preservatives<br>A1CD (Hemolyzing Reagent):<br>• Aqueous buffered matrix, pH 7.25<br>• Tetradecyltrimethylammonium bromide: 36<br>g/L<br>• sodium dihydrogenphosphate monohydrate:<br>16 mmol/L<br>• sodium monohydrogenphosphate dihydrate:<br>64 mmol/L<br>stabilizers; preservatives | R1 Antibody Reagent:<br>•MES (2-morpholinoethane sulfonic<br>acid) buffer: 0.025 mol/L<br>•TRIS (Tris(hydroxymethyl)aminomethane)<br>buffer: 0.015 mol/L, pH 6.2<br>•HbA1c antibody (ovine serum): ≥ 0.5 mg/ml<br>• detergents; stabilizers; preservatives<br>R3 Polyhapten Reagent:<br>•MES buffer: 0.025 mol/L<br>•TRIS buffer: 0.015 mol/L, pH 6.2<br>•HbA1c polyhapten: > 8 µg/mL<br>• detergents; stabilizers; preservatives<br>A1CD (Hemolyzing Reagent):<br>• Aqueous buffered matrix, pH 7.25<br>• Tetradecyltrimethylammonium bromide: 36<br>g/L<br>• Sodium dihydrogenphosphate monohydrate:<br>16 mmol/L<br>• Sodium monohydrogenphosphate dihydrate:<br>64 mmol/L<br>stabilizers; preservatives |

Table 1: Substantial Equivalence Assay Comparison

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#### 4. NON-CLINICAL PERFORMANCE EVALUATION

Performance characteristics were evaluated with Tina-quant Hemoglobin A1cDx Gen.3 on the cobas c 503 analytical unit.

Tina-quant Hemoglobin A1cDx Gen.3 offers two sample type specific applications, Hemolysate Application and Whole Blood Application, one for manually hemolyzed samples and one for whole blood samples respectively.

The Tina-quant Hemoglobin A1cDx Gen.3 assay first measures total hemoglobin (Hb) and glycated hemoglobin (HbA1c) in terms of either g/dL or mmol/L. Then the analyzer calculates the HbA1c/Hb ratio according to either IFCC or DCCT/NGSP. IFCC protocol reports the ratio in terms of mmol/mol HbA1c while the DCCT/NGSP protocol reports the ratio in terms of % HbA1c. Performance characteristics that support the measuring ranges claimed for Hb and HbA1c included limit of detection and linearity. These results were reported in terms of Hb and HbA1c individually. Patient sample values are reported in terms of the ratio of glycated to total hemoglobin. Method comparison, control recovery and precision were evaluated in terms of the ratio.

The following performance data were provided in support of the substantial equivalence determination:

#### Precision 4.1.

#### Repeatability and Intermediate Precision 4.1.1.

Precision measurements were conducted to evaluate repeatability (within-run precision) and intermediate precision (within-laboratory precision) according the CLSI guideline EP5-A3.

Two aliquots per sample were measured once each, in two runs per day, for 21 days, on 3 cobas c 503 analytical units and using 3 reagent lots per system. Ten total samples were evaluated in each run: two controls, PreciControl HbA1c norm and PreciControl HbA1c path, and eight human samples with approximate Hb1Ac concentrations of 4.9%, 6.6%, 7.3%, 8.2%, 12.5%, 14.6%, 12.3% and 13.1% for Whole Blood and 5.0%, 6.6%, 7.3%, 8.3%, 12.5%, 14.7%, 12.1% and 12.9% for Hemolysate.

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The samples were randomized within each run. For each sample, the following was calculated: mean, repeatability and intermediate precision as CV and SD values and the upper 95% confidence interval for SD and CV values.

| Mean<br>% HbA1c | Repeatability (error) | | Between-Run | | Between-Day | | Between-lot | | Intermediate Precision<br>(total) | |
|-----------------|-----------------------|--------|-------------|--------|-------------|--------|-------------|--------|-----------------------------------|--------|
| | SD | CV (%) | SD | CV (%) | SD | CV (%) | SD | CV (%) | SD | CV (%) |
| Hem 1<br>4.96 | 0.025 | 0.5 | 0.006 | 0.1 | 0.071 | 1.4 | 0.016 | 0.3 | 0.077 | 1.6 |
| Hem 2<br>6.62 | 0.027 | 0.4 | 0.013 | 0.2 | 0.053 | 0.8 | 0.059 | 0.9 | 0.085 | 1.3 |
| Hem 3<br>7.32 | 0.035 | 0.5 | 0.000 | 0.0 | 0.053 | 0.7 | 0.067 | 0.9 | 0.092 | 1.3 |
| Hem 4<br>8.32 | 0.039 | 0.5 | 0.009 | 0.1 | 0.056 | 0.7 | 0.083 | 1.0 | 0.108 | 1.3 |
| Hem 5<br>12.54 | 0.057 | 0.5 | 0.011 | 0.1 | 0.100 | 0.8 | 0.203 | 1.6 | 0.234 | 1.9 |
| HE 006<br>14.77 | 0.077 | 0.5 | 0.013 | 0.1 | 0.148 | 1.0 | 0.268 | 1.8 | 0.316 | 2.1 |
| HE_007<br>12.14 | 0.055 | 0.5 | 0.023 | 0.2 | 0.100 | 0.8 | 0.181 | 1.5 | 0.215 | 1.8 |
| HE_008<br>12.94 | 0.072 | 0.6 | 0.000 | 0.0 | 0.111 | 0.9 | 0.188 | 1.5 | 0.230 | 1.8 |
| PCA1N<br>5.53 | 0.024 | 0.4 | 0.009 | 0.2 | 0.059 | 1.1 | 0.028 | 0.5 | 0.071 | 1.3 |
| PCA1P<br>10.89 | 0.055 | 0.5 | 0.026 | 0.2 | 0.085 | 0.8 | 0.146 | 1.3 | 0.179 | 1.6 |

Table 2: Precision Results – Hemolysate Application, Analytical Unit 1

| Table 3: Precision Results - Hemolysate Application, Analytical Unit 2 | | | |
|------------------------------------------------------------------------|--|--|--|
| | | | |

| Mean<br>% HbA1c | | Repeatability (error) | Between-Run | | Between-Day | | | Between-lot | Intermediate Precision<br>(total) | |
|-----------------|-------|-----------------------|-------------|--------|-------------|--------|-------|-------------|-----------------------------------|--------|
| | SD | CV (%) | SD | CV (%) | SD | CV (%) | SD | CV (%) | SD | CV (%) |
| Hem 1<br>4.96 | 0.027 | 0.5 | 0.005 | 0.1 | 0.034 | 0.7 | 0.015 | 0.3 | 0.046 | 0.9 |
| Hem 2<br>6.59 | 0.035 | 0.5 | 0.000 | 0.0 | 0.038 | 0.6 | 0.057 | 0.9 | 0.077 | 1.2 |
| Hem 3<br>7.29 | 0.041 | 0.6 | 0.000 | 0.0 | 0.043 | 0.6 | 0.068 | 0.9 | 0.090 | 1.2 |
| Hem 4<br>8.28 | 0.039 | 0.5 | 0.015 | 0.2 | 0.046 | 0.6 | 0.093 | 1.1 | 0.112 | 1.4 |
| Hem 5<br>12.43 | 0.069 | 0.6 | 0.027 | 0.2 | 0.038 | 0.3 | 0.175 | 1.4 | 0.193 | 1.6 |
| HE 006<br>14.68 | 0.085 | 0.6 | 0.011 | 0.1 | 0.060 | 0.4 | 0.220 | 1.5 | 0.243 | 1.7 |
| HE_007<br>12.05 | 0.063 | 0.5 | 0.018 | 0.1 | 0.036 | 0.3 | 0.163 | 1.4 | 0.179 | 1.5 |
| HE_008<br>12.85 | 0.071 | 0.6 | 0.034 | 0.3 | 0.053 | 0.4 | 0.177 | 1.4 | 0.201 | 1.6 |
| PCA1N<br>5.52 | 0.030 | 0.5 | 0.008 | 0.1 | 0.029 | 0.5 | 0.024 | 0.4 | 0.049 | 0.9 |
| PCA1P<br>10.81 | 0.074 | 0.7 | 0.000 | 0.0 | 0.041 | 0.4 | 0.134 | 1.2 | 0.159 | 1.5 |

# Table 4: Precision Results – Hemolysate Application, Analytical Unit 3

| Mean %<br>HbA1c | Repeatability (error)<br>SD | Repeatability (error)<br>CV (%) | Between-Run<br>SD | Between-Run<br>CV (%) | Between-Day<br>SD | Between-Day<br>CV (%) | Between-lot<br>SD | Between-lot<br>CV (%) | Intermediate Precision (total)<br>SD | Intermediate Precision (total)<br>CV (%) |
|-----------------|-----------------------------|---------------------------------|-------------------|-----------------------|-------------------|-----------------------|-------------------|-----------------------|--------------------------------------|------------------------------------------|
| Hem 1<br>4.94 | 0.027 | 0.5 | 0.010 | 0.2 | 0.031 | 0.6 | 0.023 | 0.5 | 0.048 | 1.0 |
| Hem 2<br>6.57 | 0.030 | 0.5 | 0.004 | 0.1 | 0.035 | 0.5 | 0.049 | 0.7 | 0.067 | 1.0 |

{11}------------------------------------------------

| Hem 3<br>7.28 | 0.032 | 0.4 | 0.000 | 0.0 | 0.038 | 0.5 | 0.054 | 0.7 | 0.073 | 1.0 |
|-----------------|-------|-----|-------|-----|-------|-----|-------|-----|-------|-----|
| Hem 4<br>8.28 | 0.040 | 0.5 | 0.000 | 0.0 | 0.041 | 0.5 | 0.070 | 0.8 | 0.091 | 1.1 |
| Hem 5<br>12.43 | 0.063 | 0.5 | 0.024 | 0.2 | 0.045 | 0.4 | 0.162 | 1.3 | 0.181 | 1.5 |
| HE 006<br>14.68 | 0.075 | 0.5 | 0.000 | 0.0 | 0.050 | 0.3 | 0.240 | 1.6 | 0.256 | 1.7 |
| HE 007<br>12.07 | 0.064 | 0.5 | 0.000 | 0.0 | 0.039 | 0.3 | 0.146 | 1.2 | 0.164 | 1.4 |
| HE_008<br>12.84 | 0.072 | 0.6 | 0.000 | 0.0 | 0.052 | 0.4 | 0.150 | 1.2 | 0.174 | 1.4 |
| PCA1N<br>5.49 | 0.026 | 0.5 | 0.008 | 0.2 | 0.035 | 0.6 | 0.024 | 0.4 | 0.050 | 0.9 |
| PCA1P<br>10.78 | 0.071 | 0.7 | 0.000 | 0.0 | 0.048 | 0.4 | 0.116 | 1.1 | 0.144 | 1.3 |

| Table 5: Precision Results - Hemolysate Application, 3 Lots and 3 Analytical Units | |
|------------------------------------------------------------------------------------|--|
|------------------------------------------------------------------------------------|--|

| Mean,<br>%HbA1c | | Repeatability<br>(error) | | Between-Run | | Between-Day | | Between-Lot | | Between-Device | | Reproducibility<br>(total) | |
|------------------|-------|--------------------------|-------|-------------|-------|-------------|-------|-------------|-------|----------------|-------|----------------------------|--|
| | SD | CV(%) | SD | CV(%) | SD | CV(%) | SD | CV(%) | SD | CV(%) | SD | CV(%) | |
| Hem 1,<br>4.96 | 0.026 | 0.5 | 0.007 | 0.1 | 0.049 | 1.0 | 0.019 | 0.4 | 0.009 | 0.2 | 0.059 | 1.2 | |
| Hem 2,<br>6.59 | 0.031 | 0.5 | 0.006 | 0.1 | 0.042 | 0.6 | 0.055 | 0.8 | 0.023 | 0.3 | 0.080 | 1.2 | |
| Hem 3,<br>7.30 | 0.036 | 0.5 | 0.000 | 0.0 | 0.045 | 0.6 | 0.063 | 0.9 | 0.019 | 0.3 | 0.088 | 1.2 | |
| Hem 4,<br>8.29 | 0.039 | 0.5 | 0.005 | 0.1 | 0.049 | 0.6 | 0.082 | 1.0 | 0.019 | 0.2 | 0.105 | 1.3 | |
| Hem 5,<br>12.47 | 0.063 | 0.5 | 0.022 | 0.2 | 0.070 | 0.6 | 0.179 | 1.4 | 0.061 | 0.5 | 0.212 | 1.7 | |
| HE_006,<br>14.71 | 0.079 | 0.5 | 0.010 | 0.1 | 0.098 | 0.7 | 0.242 | 1.6 | 0.053 | 0.4 | 0.278 | 1.9 | |
| HE_007,<br>12.08 | 0.061 | 0.5 | 0.016 | 0.1 | 0.067 | 0.6 | 0.163 | 1.3 | 0.048 | 0.4 | 0.193 | 1.6 | |
| HE_008,<br>12.88 | 0.072 | 0.6 | 0.017 | 0.1 | 0.078 | 0.6 | 0.172 | 1.3 | 0.055 | 0.4 | 0.210 | 1.6 | |
| PCA1N,<br>5.51 | 0.027 | 0.5 | 0.008 | 0.2 | 0.043 | 0.8 | 0.027 | 0.5 | 0.022 | 0.4 | 0.062 | 1.1 | |
| PCA1P,<br>10.83 | 0.067 | 0.6 | 0.000 | 0.0 | 0.062 | 0.6 | 0.132 | 1.2 | 0.054 | 0.5 | 0.169 | 1.6 | |

# Table 6: Precision Results – Whole Blood Application, Analytical Unit 1

| Mean | Repeatability (error) | | Between-Run | | Between-Day | | Between-Lot | | Intermediate Precision<br>(total) | |
|------------------|-----------------------|-------|-------------|-------|-------------|-------|-------------|-------|-----------------------------------|-------|
| %HbA1c | SD | CV(%) | SD | CV(%) | SD | CV(%) | SD | CV(%) | SD | CV(%) |
| WB 1,<br>4.87 | 0.038 | 0.8 | 0.000 | 0.0 | 0.043 | 0.9 | 0.045 | 0.9 | 0.073 | 1.5 |
| WB 2,<br>6.60 | 0.026 | 0.4 | 0.015 | 0.2 | 0.030 | 0.4 | 0.071 | 1.1 | 0.083 | 1.3 |
| WB 3,<br>7.37 | 0.032 | 0.4 | 0.018 | 0.2 | 0.032 | 0.4 | 0.084 | 1.1 | 0.097 | 1.3 |
| WB 4,<br>8.24 | 0.039 | 0.5 | 0.008 | 0.1 | 0.048 | 0.6 | 0.089 | 1.1 | 0.108 | 1.3 |
| WB 5,<br>12.59 | 0.056 | 0.4 | 0.025 | 0.2 | 0.061 | 0.5 | 0.154 | 1.2 | 0.177 | 1.4 |
| WB_006,<br>14.69 | 0.079 | 0.5 | 0.043 | 0.3 | 0.067 | 0.5 | 0.168 | 1.1 | 0.202 | 1.4 |
| WB_007,<br>12.34 | 0.062 | 0.5 | 0.032 | 0.3 | 0.057 | 0.5 | 0.152 | 1.2 | 0.176 | 1.4 |
| WB_008,<br>13.14 | 0.061 | 0.5 | 0.015 | 0.1 | 0.055 | 0.4 | 0.165 | 1.3 | 0.185 | 1.4 |
| PCA1N,<br>5.51 | 0.029 | 0.5 | 0.002 | 0.0 | 0.039 | 0.7 | 0.044 | 0.8 | 0.066 | 1.2 |
| PCA1P,<br>11.20 | 0.055 | 0.5 | 0.011 | 0.1 | 0.057 | 0.5 | 0.129 | 1.2 | 0.152 | 1.4 |

{12}------------------------------------------------

| Mean<br>%HbA1c | Repeatability (error) | | Between-Run | | Between-Day | | Between-Lot | | Intermediate Precision<br>(total) | |
|------------------|-----------------------|-------|-------------|-------|-------------|-------|-------------|-------|-----------------------------------|-------|
| | SD | CV(%) | SD | CV(%) | SD | CV(%) | SD | CV(%) | SD | CV(%) |
| WB 1,<br>4.88 | 0.032 | 0.7 | 0.013 | 0.3 | 0.026 | 0.5 | 0.031 | 0.6 | 0.053 | 1.1 |
| WB 2,<br>6.58 | 0.031 | 0.5 | 0.010 | 0.2 | 0.025 | 0.4 | 0.077 | 1.2 | 0.087 | 1.3 |
| WB 3,<br>7.35 | 0.040 | 0.5 | 0.000 | 0.0 | 0.029 | 0.4 | 0.091 | 1.2 | 0.103 | 1.4 |
| WB 4,<br>8.21 | 0.042 | 0.5 | 0.011 | 0.1 | 0.035 | 0.4 | 0.106 | 1.3 | 0.120 | 1.5 |
| WB 5,<br>12.53 | 0.070 | 0.6 | 0.029 | 0.2 | 0.047 | 0.4 | 0.164 | 1.3 | 0.186 | 1.5 |
| WB_006,<br>14.62 | 0.095 | 0.6 | 0.035 | 0.2 | 0.091 | 0.6 | 0.198 | 1.4 | 0.240 | 1.6 |
| WB_007,<br>12.25 | 0.078 | 0.6 | 0.000 | 0.0 | 0.035 | 0.3 | 0.176 | 1.4 | 0.195 | 1.6 |
| WB_008,<br>13.06 | 0.073 | 0.6 | 0.009 | 0.1 | 0.052 | 0.4 | 0.163 | 1.2 | 0.186 | 1.4 |
| PCA1N,<br>5.51 | 0.036 | 0.6 | 0.000 | 0.0 | 0.028 | 0.5 | 0.044 | 0.8 | 0.063 | 1.1 |
| PCA1P,<br>11.13 | 0.062 | 0.6 | 0.033 | 0.3 | 0.043 | 0.4 | 0.162 | 1.5 | 0.182 | 1.6 |

Table 7: Precision Results – Whole Blood Application, Analytical Unit 2

| | | | | | Table 8: Precision Results - Whole Blood Application, Analytical Unit 3 | | |
|--|--|--|--|--|--------------------------------------------------------------------------|--|--|
|--|--|--|--|--|--------------------------------------------------------------------------|--|--|

| Mean | Repeatability (error) | | Between-Run | | Between-Day | | Between-Lot | | Intermediate Precision<br>(total) | |
|------------------|-----------------------|-------|-------------|-------|-------------|-------|-------------|-------|-----------------------------------|-------|
| %HbA1c | SD | CV(%) | SD | CV(%) | SD | CV(%) | SD | CV(%) | SD | CV(%) |
| WB 1,<br>4.86 | 0.029 | 0.6 | 0 | 0.0 | 0.035 | 0.7 | 0.044 | 0.9 | 0.064 | 1.3 |
| WB 2,<br>6.55 | 0.031 | 0.5 | 0.009 | 0.1 | 0.025 | 0.4 | 0.080 | 1.2 | 0.090 | 1.4 |
| WB 3,<br>7.31 | 0.039 | 0.5 | 0.006 | 0.1 | 0.026 | 0.4 | 0.090 | 1.2 | 0.101 | 1.4 |
| WB 4,<br>8.17 | 0.043 | 0.5 | 0.012 | 0.2 | 0.036 | 0.4 | 0.096 | 1.2 | 0.111 | 1.4 |
| WB 5,<br>12.51 | 0.079 | 0.6 | 0.000 | 0.0 | 0.064 | 0.5 | 0.154 | 1.2 | 0.184 | 1.5 |
| WB_006,<br>14.62 | 0.082 | 0.6 | 0.000 | 0.0 | 0.107 | 0.7 | 0.191 | 1.3 | 0.234 | 1.6 |
| WB_007,<br>12.23 | 0.068 | 0.6 | 0.028 | 0.2 | 0.056 | 0.5 | 0.157 | 1.3 | 0.182 | 1.5 |
| WB_008,<br>13.05 | 0.083 | 0.6 | 0.000 | 0.0 | 0.064 | 0.5 | 0.162 | 1.2 | 0.193 | 1.5 |
| PCA1N,<br>5.50 | 0.030 | 0.6 | 0.006 | 0.1 | 0.028 | 0.5 | 0.051 | 0.9 | 0.066 | 1.2 |
| PCA1P,<br>11.10 | 0.071 | 0.6 | 0.000 | 0.0 | 0.043 | 0.4 | 0.137 | 1.2 | 0.160 | 1.4 |

{13}------------------------------------------------

| Mean | | Repeatability<br>(error) | | Between-Run | | Between-Day | | Between-Lot | | Between-Device | | Reproducibility<br>(total) | |
|------------------|-------|--------------------------|-------|-------------|-------|-------------|-------|-------------|-------|----------------|-------|----------------------------|--|
| %HbA1c | SD | CV(%) | SD | CV(%) | SD | CV(%) | SD | CV(%) | SD | CV(%) | SD | CV(%) | |
| WB 1,<br>4.87 | 0.034 | 0.7 | 0.004 | 0.1 | 0.036 | 0.7 | 0.040 | 0.8 | 0.007 | 0.1 | 0.064 | 1.3 | |
| WB 2,<br>6.57 | 0.029 | 0.4 | 0.012 | 0.2 | 0.028 | 0.4 | 0.075 | 1.1 | 0.024 | 0.4 | 0.090 | 1.4 | |
| WB 3,<br>7.34 | 0.037 | 0.5 | 0.009 | 0.1 | 0.032 | 0.4 | 0.087 | 1.2 | 0.027 | 0.4 | 0.104 | 1.4 | |
| WB 4,<br>8.20 | 0.041 | 0.5 | 0.011 | 0.1 | 0.042 | 0.5 | 0.096 | 1.2 | 0.031 | 0.4 | 0.117 | 1.4 | |
| WB 5,<br>12.54 | 0.069 | 0.5 | 0.020 | 0.2 | 0.062 | 0.5 | 0.155 | 1.2 | 0.043 | 0.3 | 0.187 | 1.5 | |
| WB_006,<br>14.64 | 0.086 | 0.6 | 0.028 | 0.2 | 0.096 | 0.7 | 0.181 | 1.2 | 0.043 | 0.3 | 0.228 | 1.6 | |
| WB_007,<br>12.27 | 0.070 | 0.6 | 0.023 | 0.2 | 0.055 | 0.5 | 0.159 | 1.3 | 0.055 | 0.4 | 0.192 | 1.6 | |
| WB_008,<br>13.08 | 0.073 | 0.6 | 0.000 | 0.0 | 0.061 | 0.5 | 0.161 | 1.2 | 0.049 | 0.4 | 0.194 | 1.5 | |
| PCA1N,<br>5.51 | 0.032 | 0.6 | 0.002 | 0.0 | 0.032 | 0.6 | 0.047 | 0.8 | 0.007 | 0.1 | 0.065 | 1.2 | |
| PCA1P,<br>11.14 | 0.063 | 0.6 | 0.019 | 0.2 | 0.054 | 0.5 | 0.141 | 1.3 | 0.047 | 0.4 | 0.171 | 1.5 | |

Table 9: Precision Results – Whole Blood Application, 3 Lots and 3 Analytical Units

#### Analytical Sensitivity 4.2.

#### Limit of Blank (LoB) 4.2.1.

For determination of LoB, one analyte free sample was measured with three lots of Tina-quant Hemoglobin A1cDx Gen.3, in 10-fold determinations. Six runs were distributed over ≥ three days and measured using one cobas c 503 analytical unit. In total, 60 measurements were obtained per lot. Data analysis was based on determination of the 95th percentile of the 60 measured values. In our design (n=60) the 95th percentile is the average of the 57th and 58th value.

LoB was determined according to CLSI guideline EP17-A2.

Table 10: LoB Results

| | Hb LoB | | HbA1c LoB | |
|--|---------------|------------|---------------|------------|
| | mmol/L | g/dL | mmol/L | g/dL |
| | 0.0530 mmol/L | 0.085 g/dL | 0.0220 mmol/L | 0.035 g/dL |

This corresponds to a Limit of Blank of 15 mmol/mol (IFCC) and 3.5 % HbA1c (DCCT/NGSP) at a typical hemoglobin concentration of 13.2 g/dL (8.2 mmol/L).

{14}------------------------------------------------

#### Limit of Detection (LoD) 4.2.2.

For determination of LoD, five unique human samples with low-analyte concentrations were measured with three lots of Tina-quant Hemoglobin A1cDx Gen.3 in two-fold determinations. The measurements were performed in six runs, over > three days, on one cobas c 503 analytical unit. In total. 60 measurements were obtained per lot.

LoD is defined as the concentration, at which there is a 95% probability that a sample contains analyte.

LoD was determined according to CLSI guideline EP17-A2.

### Table 11: LoD Results

| Hb LoD | | HbA1c LoD | |
|--------------|------------|---------------|-----------|
| mmol/L | g/dL | mmol/L | g/dL |
| 0.119 mmol/L | 0.192 g/dL | 0.0437 mmol/L | 0.07 g/dL |

This corresponds to a Limit of Detection of 22 mmol/mol (IFCC) and 4.2 % HbA1c (DCCT/NGSP) at a typical hemoglobin concentration of 13.2 g/dL (8.2 mmol/L).

#### Linearity/Assay Reportable Range 4.3.

Separate dilution series, consisting of at least eleven levels, were prepared for each glycated hemoglobin (HbA1c) and total hemoglobin (Hb) using human hemolysate sample pools. The sample pools include HbA1c and Hb concentrations above the upper end of the corresponding measuring range. Hemolyzing reagent was used for the diluent. Samples were measured in triplicate and data analysis was performed separately for each sample.

The study was performed according to CLSI guideline EP6-A.

Table 12: Linearity Results according to CLSI EP6-A

| Application | Analyte | Low End of Linear Range | | High End of Linear Range | |
|-------------|---------|-------------------------|--------|--------------------------|--------|
| | | g/dL | mmol/L | g/dL | mmol/L |
| Hemolysate | Hb | 3.04 | 1.89 | 40.4 | 25.1 |
| | HbA1c | 0.293 | 0.182 | 2.87 | 1.78 |

{15}------------------------------------------------

Table 13: Empirical First Order Regression Results

| Application | Analyte | Slope | Intercept | Pearson's r |
|-------------|---------|-------|-----------|-------------|
| Hemolysate | Hb | 1.019 | -0.1552 | 0.9999 |
| | HbA1c | 0.991 | -0.0026 | 0.9990 |

Linearity was determined throughout the claimed measuring range of:

4 – 40 g/dL (2.48 – 24.8 mmol/L) Hb:

0.3 – 2.6 g/dL (0.186 – 1.61 mmol/L) HbA1c:

This corresponds to a measuring range of 23-196 mmol/mol HbA1c (IFCC) and 4.2-20.1 % HbA1c (DCCT/NGSP) at a typical hemoglobin concentration of 13.2 g/dL (8.2 mmol/L).

#### 4.4. Endogenous Interferences

A study evaluated several endogenous substances for potential interference with measurement of % HbA1c. The following nine endogenous substances were evaluated.

- Bilirubin .
- Ditaurobilirubin .
- Lipemia .
- Rheumatoid Factors .
- Total Protein .
- Albumin .
- Immunoglubulin (IgG) .
- Glucose .
- Triglycerides .

Pooled whole blood samples, with two hemoglobin A1c levels, one near the medical decision level and one above it, were spiked with the maximum level of the above nine interferents, in separate preparations, resulting in eighteen spiked samples were then hemolyzed

{16}------------------------------------------------

with Tina-quant HbA1c Hemolyzing Reagent. Another pool, without interferent, was equally hemolyzed. A > ten-level dilution series was created for each of the eighteen spiked samples, using the interferent-free pools as the diluent.

The eighteen dilution series were tested in ten-fold, using one reagent lot, one cobas c 503 analytical unit, in a single run and within one calibration cycle. Additionally, PreciControl HbA1c norm and PreciControl HbA1c path were used as the controls. The mean of the ten replicates was compared to the result from the reference sample (aliquot with no interferent). The comparison was evaluated as a percent deviation. For purposes of this experiment, the data was collected using the Hemolysate Application and was representative of both Hemolysate and Whole Blood Applications.

| Potential Interferent | Claimed Maximum Concentration without Interference |
|-----------------------|----------------------------------------------------|
| Bilirubin | 60 mg/dL |
| Ditaurobilirubin | 60 mg/dL |
| Lipemia | 400 mg/dL |
| Rheumatoid Factors | 750 IU/mL |
| Total Protein | 21 g/dL |
| Albumin | 60 g/L |
| Immunoglobulin (IgG) | 60 g/L |
| Glucose | 1000 mg/dL |
| Triglycerides | 1584 mg/dL |

## Table 14: Endogenous Interference

#### 4.5. Cross-Reactivity

This study was conducted to evaluate the Tina-quant Hemoglobin A1cDx Gen.3 assay on the cobas c 503 analytical unit for potential cross-reactivity with the following hemoglobin fractions and glycated albumin.

- HbA0 .
- Carbamylated Hb .
- HbA1(a+b) .
- Acetylated Hb .
- Labile HbA1c Glycated Albumin . .

{17}------------------------------------------------

A series of experiments were performed using one reagent lot, on one cobas c 503 analytical unit, in a single run, within one calibration cycle. PreciControl HbA1c norm and PreciControl HbA1c path were used as the controls. Ten replicates of each sample were analyzed for each dilution level. The median % HbA1c of each dilution level was compared to the median % HbA1c from dilution level zero (without cross-reactant). For purposes of this experiment, the data was collected using the Hemolysate Application and was representative of both Hemolysate and Whole Blood Applications.

| Cross-Reactant | | Max Whole Blood Cross-<br>Reactant Concentration | Max Whole Blood Cross-<br>Reactant Concentration with<br>no Interference |
|------------------|---------------|--------------------------------------------------|--------------------------------------------------------------------------|
| HbA0 | HbA1c Level 1 | 120 g/dL | 120 g/dL |
| | HbA1c Level 2 | 120 g/dL | 120 g/dL |
| HbA1(a+b) | HbA1c Level 1 | 1.6 g/dL | 0.96 g/dL |
| | HbA1c Level 2 | 1.6 g/dL | 1.6 g/dL |
| Carbamylated Hb | HbA1c Level 1 | 2.0 g/dL | 2.0 g/dL |
| | HbA1c Level 2 | 2.0 g/dL | 2.0 g/dL |
| Acetylated Hb | HbA1c Level 1 | 2.0 g/dL | 2.0 g/dL |
| | HbA1c Level 2 | 2.0 g/dL | 2.0 g/dL |
| Glycated Albumin | HbA1c Level 1 | 10 g/dL | 10 g/dL |
| | HbA1c Level 2 | 10 g/dL | 10 g/dL |
| Labile HbA1c | HbA1c Level 1 | 1000 mg/dL | 1000 mg/dL |
| | HbA1c Level 2 | 1000 mg/dL | 1000 mg/dL |

Table 15: Cross-Reactivity

#### Hemoglobin Variants 4.6.

Hemoglobin variant testing was conducted to determine if significant interference with any of the major hemoglobin variants occurred when using the Tina-quant Hemoglobin A1cDx Gen.3 assay on cobas c 503 analytical unit. Hemoglobin variants are structurally altered hemoglobin molecules with at least one amino acid exchange, compared to the normal beta chain of hemoglobin. These changes are caused by mutations in the coding region of the globin genes which encode the protein part of hemoglobin. The most common hemoglobin variants are HbS, HbC, HbD and HbE. Additionally, in some conditions fetal hemoglobin, HbF, is elevated. Also, the erythrocytes of some patients (e.g. beta thalassemia minor) contain elevated levels of HbA2.

{18}------------------------------------------------

Therefore, it is crucial to ensure accurate HbA1c results from patients who are carriers of these variants.

| Variant Type | Number of Samples | % Variant | HbA1c % |
|--------------|-------------------|-------------|-------------|
| HbS | 30 | 35-41% S | 4.35 - 12.7 |
| HbC | 30 | 28-37% C | 4.90 - 14.1 |
| HbE | 30 | 24-27% E | 5.17 - 10.0 |
| HbD | 29 | 36-42% D | 5.17 - 9.70 |
| HbA2 | 15 | 4.3-6.5% A2 | 5.10 - 9.80 |
| Elevated HbF | 19 | 3.2-39% F | 6.10 - 9.30 |

### Table 16: Hemoglobin Variant Samples

Each sample was tested once, in at least one run, on one cobas c 503 analytical unit. Results obtained with the Tina-quant Hemoglobin A1cDx Gen.3 assay on the cobas c 503 analytical unit were compared to those obtained with the corresponding reference method. For purposes of this experiment, the data was collected using the Hemolysate Application and was representative of both Hemolysate and Whole Blood Applications.

Table 17: Hemoglobin Variant Testing

| Percent Relative Bias from Reference Method at Low and High Concentrations of HbA1c Samples | | | | |
|---------------------------------------------------------------------------------------------|--------------------------------------------------------------------------------------------|------------|-----------------------|---------------|
| | Around 6.5% HbA1c | | Around 9% HbA1c | |
| HbVariant | Relative % Difference | Range | Relative % Difference | Range |
| HbS | -2.5 | -7.2 - 3.2 | -4.0 | -9.3 - (-2.0) |
| HbC | -3.9 | -7.7 - 2.8 | -6.0 | -4.6 - (-3.6) |
| HbE | -0.1 | -5.5 - 5.7 | -1.2 | -5.2 - 0.6 |
| HbD | -1.8 | -4.5 - 3.0 | -2.6 | -3.3 - 0.2 |
| HbA2 | -1.0 | -4.1 - 2.7 | 0.4 | -2.2 - 1.1 |
| HbF | Specimens containing high amounts of HbF (>7%) may yield lower than expected HbA1c values. | | | |

#### Exogenous Interferences – Drugs 4.7.

The purpose of this study was to evaluate drugs for potential interference with the Tina-quant Hemoglobin A1cDx Gen.3 assay measured on the cobas c 503 analytical u…