VARIANT II TURBO HbA1c Kit - 2.0

K142448 · Bio-Rad Laboratories, Inc. · PDJ · Mar 24, 2015 · Clinical Chemistry

Device Facts

• Record ID: K142448
• Device Name: VARIANT II TURBO HbA1c Kit - 2.0
• Applicant: Bio-Rad Laboratories, Inc.
• Product Code: PDJ · Clinical Chemistry
• Decision Date: Mar 24, 2015
• Decision: SESE
• Submission Type: Traditional
• Regulation: 21 CFR 862.1373
• Device Class: Class 2

Indications for Use

The VARIANT™ II TURBO HbA1c Kit- 2.0 is intended for the quantitative determination of hemoglobin A1c (IFCC mmol/mol and NGSP %) in human whole blood using ion-exchange high-performance liquid chromatography (HPLC) on the VARIANT II TURBO Hemoglobin Testing System and VARIANT II TURBO Link Hemoglobin Testing System. This test is to be used as an aid in diagnosis of diabetes and as an aid in identifying patients who may be at risk for developing diabetes. The VARIANT II TURBO HbA1c Kit- 2.0 is intended for Professional Use Only. The Hemoglobin Capillary Collection System (HCCS) is intended for the collection of human whole blood for the percent determination of hemoglobin A1c using Bio-Rad HPLC methods.

Device Story

The VARIANT™ II TURBO HbA1c Kit- 2.0 is an in vitro diagnostic assay for professional use in clinical laboratories. It utilizes ion-exchange high-performance liquid chromatography (HPLC) to separate hemoglobins in human whole blood samples. Samples are automatically diluted by the VARIANT™ II TURBO Sampling Station (VSS) and injected into an analytical cartridge. A programmed buffer gradient of increasing ionic strength separates hemoglobins based on ionic interactions. Separated hemoglobins pass through a filter photometer measuring absorbance at 415 nm, with 690 nm used for background correction. The Clinical Data Management (CDM™) software processes raw data, calculates HbA1c area using an exponentially modified Gaussian (EMG) algorithm, and generates reports. Results are used by clinicians to monitor long-term glycemic control and diagnose diabetes. The system benefits patients by providing standardized, precise HbA1c measurements traceable to DCCT and IFCC reference methods.

Clinical Evidence

Bench testing only. Precision evaluated per CLSI EP05-A2 (n=720 measurements per sample). Linearity demonstrated across 3.4–20.6% HbA1c range (R²=0.998). Method comparison (n=130) against NGSP Secondary Reference Laboratory showed Deming slope 1.033 and intercept -0.275. Interference studies confirmed no significant impact from endogenous substances, drugs, or hemoglobin variants (S, C, E, D, A2, F). Total Error calculated at 2.6%–5.2% across clinical decision points.

Technological Characteristics

Ion-exchange HPLC; dual-pump buffer gradient system; filter photometer (415 nm/690 nm); analytical cartridge; automated sampling station. Software-based data reduction and peak identification. Standardized to NGSP/DCCT/IFCC. Professional use only.

Indications for Use

Indicated for quantitative HbA1c determination in human whole blood to aid in diabetes diagnosis and risk assessment. Not for use in newborns, pregnant women, or patients with Type 1 diabetes. Contraindicated for patients with conditions altering red blood cell lifespan (e.g., hemolytic anemia, blood loss, transfusion, hemoglobinopathies, thalassemias), malignancies, or severe hepatic/renal disease. Not for diagnosing gestational diabetes.

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

• Roche COBAS INTEGRA 800 Tina-quant HbA1c DX Gen. 2 assay (k121291)

Submission Summary (Full Text)

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# 510(k) SUBSTANTIAL EQUIVALENCE DETERMINATION DECISION SUMMARY ASSAY ONLY TEMPLATE

A. 510(k) Number:
K142448

B. Purpose for Submission:
Addition of a diagnostic claim to an existing device

C. Measurand:
Whole Blood Glycosylated Hemoglobin (HbA1c)

D. Type of Test:
Ion-exchange high-performance liquid chromatography (HPLC)

E. Applicant:
Bio-Rad Laboratories, Inc

F. Proprietary and Established Names:
VARIANT II TURBO HbA1c Kit – 2.0
Hemoglobin Capillary Collection System

G. Regulatory Information:

| Regulatory Description | Classification | Regulation | Product Code | Panel |
| --- | --- | --- | --- | --- |
| Hemoglobin a1c Test System | II | 21 CFR 862.1373 | PDJ | Chemistry, 75 |
| Tubes, vials, systems, serum separators, blood collection | II | 21 CFR 862.1675 | JKA | Chemistry, 75 |

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H. Intended Use:

1. Intended use(s):

See indications for use below.

2. Indication(s) for use:

The VARIANT II TURBO HbA1c Kit – 2.0 is intended for the quantitative determination of hemoglobin A1c (IFCC mmol/mol and NGSP %) in human whole blood using ion-exchange high-performance liquid chromatography (HPLC) on the VARIANT™ II TURBO Hemoglobin Testing System and VARIANT II TURBO Link Hemoglobin Testing System.

This test is to be used as an aid in diagnosis of diabetes and as an aid in identifying patients who may be at risk for developing diabetes.

The VARIANT™ II TURBO HbA1c Kit – 2.0 is intended for Professional Use Only.

The Hemoglobin Capillary Collection System (HCCS) is intended for the collection of human whole blood for the percentage determination of hemoglobin A1c using Bio-Rad HPLC methods

3. Special conditions for use statement(s):

The HbA1c test is not intended for analysis of samples collected from newborns.

The HbA1c test should not be used to replace glucose testing in pediatric patients, pregnant women, or patients with Type 1 diabetes.

The HbA1c test should not be used to diagnose diabetes during pregnancy.

The HbA1c test should not be used to diagnose diabetes in patients with the following conditions:

- Any condition that alters the life span of the red blood cells, including recent blood loss, transfusion, significant iron deficiency, hemolytic anemia (including hereditary spherocytosis), or other hemolytic diseases, hemoglobinopathies and thalassemias, as the altered red blood cell turnover interferes with the relationship between mean blood glucose and HbA1c values.
- Malignancies or severe chronic hepatic and renal disease.

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- In cases of rapidly evolving type 1 diabetes the increase of HbA1c values might be delayed compared to the acute increase in glucose concentrations. In these conditions diabetes mellitus must be diagnosed based on plasma glucose concentration and/or the typical clinical symptoms.
- Hemoglobin A1c should not be used in the diagnosis of gestational diabetes.

For prescription use only.

## 4. Special instrument requirements:

All performance data was conducted using the Bio-Rad VARIANT II TURBO Hemoglobin Testing System. The VARIANT II TURBO and the VARIANT II TURBO Link Systems are identical with respect to all operational and system components (See Device Description below).

## I. Device Description:

The Bio-Rad VARIANT II TURBO HbA1c Kit – 2.0 contains the following supplies for 2500 tests:

| Quantity | Description |
| --- | --- |
| 2 each | Whole Blood Primer. Each vial contains lyophilized human red blood cell hemolysate with gentamicin, tobramycin, and EDTA as preservatives. Reconstituted volume is 1.0 mL per vial. |
| 5 each | Elution Buffer A. Each bottle contains 2500 mL of a succinate/sodium perchlorate buffer. Contains <0.05% sodium azide as a preservative. |
| 1 each | Elution Buffer B. Each bottle contains 2000 mL of a succinate/sodium perchlorate buffer. Contains <0.05% sodium azide as a preservative. |
| 1 each | Calibrator/Diluent Set. One set consisting of 2 vials of Calibrator Level 1, 2 vials of Calibrator Level 2, and 1 bottle of Calibrator Diluent. The calibrator vials contain lyophilized human red blood cell hemolysate with gentamicin, tobramycin, and EDTA as preservatives. Reconstituted volume is 7 mL per vial. Calibrator Diluent contains 100 mL of deionized water with <0.05% sodium azide as a preservative. |
| 1 each | CD with VARIANT II TURBO HbA1c Kit - 2.0 test parameters. |

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1 each
Analytical Cartridge. Cation exchange cartridge (2500 tests), 4.6 mm ID x 27.5 mm. 5 prefilters (500 tests each) are included with the cartridge.

1 each
Sample Vials. 100 polypropylene microvials with pierceable caps, 1.5 mL.

The Calibrators and the Whole Blood Primer contain lyophilized human red blood cell hemolysate with gentamicin, tobramycin, and EDTA as preservatives.

Each unit of whole blood used in the manufacture of the calibrators and whole blood primer was tested by FDA accepted methods and found non-reactive for HIV-1, HIV-2, Hepatitis B (HBV), Hepatitis C (HCV), and syphilis.

Calibrator Level 1, Calibrator Level 2 were previously cleared in k070452.

The VARIANT II TURBO HbA1c Kit – 2.0 is designed to be used on the standalone VARIANT II TURBO and the VARIANT II TURBO Link Hemoglobin Testing Systems. VARIANT II TURBO and the VARIANT II TURBO Link are identical with respect to all operational and system components. Physically, VARIANT II TURBO Link VSS outer case is modified for compatibility with a track system. In addition, the barcode reader, tube spinner and tube sensor are controlled by the line system in the VARIANT II TURBO Link Hemoglobin Testing System. Functionality on the VARIANT II TURBO Link has not changed, just the physical orientation to accommodate sample tube management.

The hemoglobin Capillary Collection System (HCCS) contains a combination of the following components:

- Sample Preparation Vials – clear microvials with blue pierceable caps, each contains 1.5 mL of HCCS reagent (aqueous solution of EDTA and potassium cyanide (.25mmol/L). The microvials are 11 mm x 40 mm and have a maximum volume of 2.0 mL.
- Capillaries – plastic capillaries (5 µL) in a dispenser
- Capillary holder – holder for manipulating the capillaries
- Labels – to label prepared samples

J. Substantial Equivalence Information:

1. Predicate device name(s):

k121291

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2. Predicate 510(k) number(s):

Roche COBAS INTEGRA 800 Tina-quant HbA1c DX Gen. 2 assay

3. Comparison with predicate:

| Similarities | | |
| --- | --- | --- |
| Item | Candidate Device: VARIANT II TURBO HbA1c Kit - 2.0 | Predicate Device: COBAS INTEGRA 800 HbA1c DX Gen. 2 |
| Intended Use | Intended for the quantitative determination of hemoglobin A1c (IFCC mmol/mol and NGSP %) in human whole blood. This test is to be used as an aid in diagnosis of diabetes and as an aid in identifying patients who may be at risk for developing diabetes. | Same |
| Measuring Range | 3.4 to 20.6 % (NSGP) 14 - 203 mmol/mol HbA1c (IFCC) | 4.3 - 24.8% (NGSP) 23 to 258 mmol/mol HbA1c (IFCC) |
| Traceability | Traceable to the Diabetes Control and Complications Trial (DCCT) reference method and IFCC. Certified via the National Glycohemoglobin Standardization Program (NGSP) | Same |

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| Differences | | |
| --- | --- | --- |
| Item | Candidate Device: VARIANT II TURBO HbA1c Kit-2.0 | Predicate Device: COBAS INTEGRA 800 HbA1c DX Gen.2 |
| Sample Types | K_{2}-EDTA, K_{3}-EDTA, Capillary blood in Hemoglobin Capillary Collection System (HCCS) | K_{2}-EDTA, K_{3}-EDTA, KF/Na2- EDTA, Na-heparin
NF/K-oxalate
NF/NA2- EDTA
Li-Heparin |
| Instrument Platform | VARIANT™ II TURBO Hemoglobin Testing System and VARIANT™ II TURBO Link Hemoglobin Testing System | Roche COBAS INTEGRA 800 analyzer |
| Assay Principal | Ion exchange HPLC | Turbidimetric inhibition immunoassay |

## K. Standard/Guidance Document Referenced (if applicable):

CLSI EP7-A2: Interference Testing in Clinical Chemistry; Approved Guideline – Second Edition

CLSI EP9-A2-IR: Method Comparison and Bias Estimation Using Patient Samples; Approved Guideline – Second Edition

CLSI EP5-A2: Evaluation of Precision Performance of Clinical Chemistry Devices; Approved Guideline

CLSI EP6-A: Evaluation of the Linearity of Quantitative Measuring Procedures: A Statistical Approach; Approved Guideline

CLSI EP14-A2: Evaluation of Matrix Effects; Approved Guideline

## L. Test Principle:

The test principal of the device is based on chromatographic separation of HbA1c on a cation exchange cartridge. The various forms of hemoglobin exhibit charge differences (positive) at the acidic pH of the mobile phase, and thus can be separated on a support that is negatively charged (cation exchange). The use of ion-exchange chromatography then allows molecules to be separated based upon a molecule's charge. Separation is optimized to minimize interferences from hemoglobin variants (HbS, HbC, HbD and HbE trait), labile A1c, hemoglobin F and carbamylated hemoglobin.

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M. Performance Characteristics (if/when applicable):

1. Analytical performance:

a. Precision/Reproducibility:

The precision of the VARIANT™ II TURBO HbA1c Kit – 2.0 was evaluated based on CLSI EP05-A2 guidelines, Evaluation of Precision Performance of Quantitative Measurement Methods using a modified study design. Four EDTA whole blood samples at the following targeted HbA1c concentrations of ~5%, ~6.5%, ~8% and ~12% were analyzed in the study. In addition, five whole blood quality control materials (Control 1, Control 2, QC1, QC2, QC3) were also tested. Precision was evaluated using three reagent lots and three VARIANT™ II TURBO Hemoglobin Testing Systems at two different sites. The samples were run in duplicate in 2 runs per day for 20 days. For each sample, there were 720 measurements. Results are shown in the tables below.

Results in NGSP Units:

Instrument 1 (% CV by Sample (NGSP))

| Variation Source | Instrument ID: VART15 | | | | | | | | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| | Control 1 | Control 2 | Patient 1 | Patient 2 | Patient 3 | Patient 4 | QC 1 | QC 2 | QC 3 |
| Concentration HbA1c (NGSP%) | 5.5 | 9.9 | 5.1 | 6.7 | 8.0 | 12.0 | 5.5 | 9.9 | 15.0 |
| Repeatability | 0.5% | 0.3% | 0.5% | 0.6% | 1.0% | 0.3% | 0.6% | 0.4% | 0.4% |
| Between-Run | 0.4% | 0.0% | 0.3% | 0.0% | 0.2% | 0.3% | 0.3% | 0.3% | 0.2% |
| Between-Day | 0.8% | 0.6% | 0.8% | 0.7% | 0.6% | 0.5% | 1.2% | 0.8% | 0.6% |
| Between-Lot | 0.8% | 0.6% | 1.0% | 0.8% | 0.6% | 0.6% | 1.0% | 0.5% | 0.2% |
| Total Precision | 1.4% | 0.9% | 1.4% | 1.2% | 1.3% | 0.9% | 1.7% | 1.0% | 0.8% |

Instrument 2 (% CV by Sample (NGSP))

| Variation Source | Instrument ID: VART17 | | | | | | | | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| | Control 1 | Control 2 | Patient 1 | Patient 2 | Patient 3 | Patient 4 | QC 1 | QC 2 | QC 3 |
| Concentration HbA1c (NGSP%) | 5.5 | 9.8 | 5.1 | 6.6 | 7.9 | 2.0 | 5.5 | 9.9 | 14.9 |
| Repeatability | 0.5% | 0.3% | 0.6% | 0.6% | 0.5% | 0.4% | 0.6% | 0.5% | 0.4% |
| Between-Run | 0.4% | 0.2% | 0.5% | 0.0% | 0.3% | 0.4% | 0.0% | 0.2% | 0.3% |
| Between-Day | 0.5% | 0.3% | 0.4% | 0.5% | 0.7% | 0.3% | 0.9% | 0.7% | 0.4% |
| Between-Lot | 0.9% | 0.7% | 0.9% | 0.7% | 0.6% | 0.4% | 1.3% | 0.6% | 0.3% |
| Total Precision | 1.2% | 0.9% | 1.3% | 1.0% | 1.1% | 0.8% | 1.7% | 1.1% | 0.8% |

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Instrument 3 (% CV by Sample (NGSP))

| Variation Source | Instrument ID: VartGerm01 | | | | | | | | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| | Control 1 | Control 2 | Patient 1 | Patient 2 | Patient 3 | Patient 4 | QC 1 | QC 2 | QC 3 |
| Concentration HbA1c (NGSP%) | 5.4 | 9.7 | 5.1 | 6.6 | 8.0 | 12.1 | 5.4 | 9.8 | 15.0 |
| Repeatability | 0.6% | 0.5% | 0.8% | 0.8% | 0.5% | 0.4% | 0.8% | 0.4% | 0.4% |
| Between-Run | 0.2% | 0.0% | 0.1% | 0.0% | 0.0% | 0.2% | 0.0% | 0.2% | 0.0% |
| Between-Day | 0.6% | 0.3% | 0.6% | 0.5% | 0.5% | 0.4% | 0.7% | 0.4% | 0.3% |
| Between-Lot | 2.0% | 0.9% | 1.6% | 1.4% | 1.0% | 0.7% | 2.2% | 1.1% | 0.7% |
| Total Precision | 2.2% | 1.1% | 1.9% | 1.7% | 1.3% | 0.9% | 2.5% | 1.2% | 0.9% |

Instruments Combined (% CV by Sample (NGSP))

| Variation Source | All Instruments | | | | | | | | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| | Control 1 | Control 2 | Patient 1 | Patient 2 | Patient 3 | Patient 4 | QC 1 | QC 2 | QC 3 |
| Concentration HbA1c (NGSP%) | 5.4 | 9.8 | 5.1 | 6.6 | 7.9 | 12.1 | 5.4 | 9.9 | 15.0 |
| Repeatability | 0.5% | 0.4% | 0.7% | 0.7% | 0.7% | 0.4% | 0.7% | 0.5% | 0.4% |
| Between-Run | 0.3% | 0.0% | 0.4% | 0.0% | 0.2% | 0.3% | 0.2% | 0.2% | 0.2% |
| Between-Day | 0.7% | 0.4% | 0.6% | 0.5% | 0.6% | 0.4% | 1.0% | 0.7% | 0.5% |
| Between-Instrument | 1.3% | 1.1% | 0.4% | 0.0% | 0.4% | 0.6% | 0.8% | 0.4% | 0.0% |
| Between-Lot | 1.4% | 0.8% | 1.2% | 1.0% | 0.8% | 0.6% | 1.6% | 0.7% | 0.5% |
| Total Precision | 2.1% | 1.5% | 1.6% | 1.3% | 1.3% | 1.1% | 2.2% | 1.2% | 0.8% |

Results in IFCC Units:

Instrument 1 (% CV by Sample (IFCC))

| Variation Source | Instrument ID: VART15 | | | | | | | | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| | Control 1 | Control 2 | Patient 1 | Patient 2 | Patient 3 | Patient 4 | QC 1 | QC 2 | QC 3 |
| Concentration HbA1c (IFCC%) | 5.5 | 9.9 | 5.1 | 6.7 | 8.0 | 12.0 | 5.5 | 9.9 | 15.0 |
| Repeatability | 0.5% | 0.3% | 0.5% | 0.6% | 1.0% | 0.3% | 0.6% | 0.4% | 0.4% |
| Between-Run | 0.4% | 0.0% | 0.3% | 0.0% | 0.2% | 0.3% | 0.4% | 0.3% | 0.2% |
| Between-Day | 0.8% | 0.6% | 0.8% | 0.7% | 0.6% | 0.5% | 1.2% | 0.8% | 0.6% |
| Between-Lot | 0.8% | 0.6% | 1.0% | 0.8% | 0.6% | 0.6% | 1.0% | 0.5% | 0.2% |
| Total Precision | 1.4% | 0.9% | 1.4% | 1.2% | 1.3% | 0.9% | 1.7% | 1.0% | 0.8% |

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Instrument 2 (% CV by Sample (IFCC))

| Variation Source | Instrument ID: VART17 | | | | | | | | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| | Control 1 | Control 2 | Patient 1 | Patient 2 | Patient 3 | Patient 4 | QC 1 | QC 2 | QC 3 |
| Concentration HbA1c (IFCC%) | 5.5 | 9.8 | 5.1 | 6.6 | 7.9 | 2.0 | 5.5 | 9.9 | 14.9 |
| Repeatability | 0.5% | 0.3% | 0.6% | 0.6% | 0.5% | 0.4% | 0.6% | 0.5% | 0.4% |
| Between-Run | 0.4% | 0.2% | 0.5% | 0.0% | 0.3% | 0.4% | 0.0% | 0.2% | 0.3% |
| Between-Day | 0.5% | 0.3% | 0.4% | 0.5% | 0.7% | 0.3% | 0.9% | 0.7% | 0.4% |
| Between-Lot | 0.9% | 0.7% | 0.9% | 0.7% | 0.6% | 0.4% | 1.3% | 0.6% | 0.3% |
| Total Precision | 1.2% | 0.9% | 1.3% | 1.0% | 1.1% | 0.8% | 1.7% | 1.1% | 0.8% |

Instrument 3 (% CV by Sample (IFCC))

| Variation Source | Instrument ID: VartGerm01 | | | | | | | | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| | Control 1 | Control 2 | Patient 1 | Patient 2 | Patient 3 | Patient 4 | QC 1 | QC 2 | QC 3 |
| Concentration HbA1c (IFCC%) | 5.4 | 9.7 | 5.1 | 6.6 | 8.0 | 12.1 | 5.4 | 9.8 | 15.0 |
| Repeatability | 0.6% | 0.5% | 0.8% | 0.8% | 0.5% | 0.4% | 0.8% | 0.4% | 0.4% |
| Between-Run | 0.2% | 0.0% | 0.1% | 0.0% | 0.0% | 0.2% | 0.0% | 0.2% | 0.0% |
| Between-Day | 0.6% | 0.3% | 0.6% | 0.5% | 0.5% | 0.4% | 0.7% | 0.4% | 0.3% |
| Between-Lot | 2.0% | 0.9% | 1.6% | 2.5% | 1.0% | 0.7% | 2.2% | 1.1% | 0.7% |
| Total Precision | 2.2% | 1.1% | 1.9% | 1.7% | 1.3% | 0.9% | 2.5% | 1.2% | 0.9% |

Instruments Combined (% CV by Sample (IFCC))

| Variation Source | All Instruments | | | | | | | | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| | Control 1 | Control 2 | Patient 1 | Patient 2 | Patient 3 | Patient 4 | QC 1 | QC 2 | QC 3 |
| Concentration HbA1c (IFCC%) | 5.4 | 9.8 | 5.1 | 6.6 | 7.9 | 12.1 | 5.4 | 9.9 | 15.0 |
| Repeatability | 0.5% | 0.4% | 0.7% | 0.7% | 0.7% | 0.4% | 0.7% | 0.5% | 0.4% |
| Between-Run | 0.3% | 0.0% | 0.4% | 0.0% | 0.2% | 0.3% | 0.2% | 0.2% | 0.2% |
| Between-Day | 0.7% | 0.4% | 0.6% | 0.5% | 0.6% | 0.4% | 1.0% | 0.7% | 0.5% |
| Between-Instrument | 1.3% | 1.1% | 0.4% | 0.0% | 0.4% | 0.6% | 0.8% | 0.4% | 0.0% |
| Between-Lot | 1.4% | 0.8% | 1.2% | 1.0% | 0.8% | 0.6% | 1.6% | 0.7% | 0.5% |
| Total Precision | 2.1% | 1.5% | 1.6% | 1.3% | 1.3% | 1.1% | 2.2% | 1.2% | 0.8% |

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# b. Linearity/assay reportable range:

A linearity study was performed per CLSI EP06-A: Evaluation of the Linearity of Quantitative Measuring Procedures; Linearity across the reportable range was performed using altered samples to obtain a low $3.4\%$ HbA1c (14 mmol/mol) and a high $20.65\%$ HbA1c (203 mmol/mol) EDTA whole blood patient samples. These samples were mixed together in varying ratios to obtain the 9 intermediate samples levels. The measured values were compared to the expected values. Polynomial regression analysis (for first, second, and third order polynomials) were performed to determine the statistical significance of non-linearity. The higher order coefficients were found not to be significant and linearity was demonstrated using the second order regression analysis. The regression parameters (slope, intercept, and $\mathbf{R}^2$ ) were the following:

NGSP:

| Slope | Intercept | R² | Sample range tested |
| --- | --- | --- | --- |
| 1.033 | -0.269 | 0.998 | 3.4-20.65% HbA1c |

IFCC:

| Slope | Intercept | R² | Sample range tested |
| --- | --- | --- | --- |
| 1.033 | -2.171 | 0.998 | 14-203 mmol/mol HbA1c |

The linearity study supports the device's claimed assay measuring range of 3.4 to $20.6\%$ HbA1c (NGSP) and 14 to $203\mathrm{mmol / mol}$ HbA1c (IFCC).

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

Traceability:

The assigned HbA1c values of the VARIANT™ II TURBO HbA1c Kit -2.0 are certified with the National Glycohemoglobin Standardization Program (NGSP). The NGSP certification expires in one year. See NGSP website for current certification at http://www.ngsp.org.

The final reportable result is traceable to both the International Federation of Clinical Chemistry (IFCC) and the Diabetes Control and Complications Trial (DCCT). The International Federation of Clinical Chemistry (IFCC) units of mmol/mol are calculated using the Master Equation NGSP $(\%) = 0.09148 \times \text{IFCC (mmol/mol)} + 2.152$ . HbA1c results are provided to the customers using two different units: NGSP equivalent units (\%) and IFCC equivalent units (mmol/mol).

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Calibrator and Control Materials:

Value assignment for calibrators (VARIANT™ II TURBO HbA1c Kit – 2.0 Calibrator/Diluent Set) which are recommended for use with this device, were previously reviewed under 510(k) submission k070452. Bio-Rad’s commercially available control materials were previously reviewed in submission k070546 and k052838.

Reagent Stability:

Reagent stability was previously reviewed in k070452. The Elution Buffers and Wash/Diluent Solution are stable until the expiration date when stored unopened at 15–30 °C. After opening the bottles, Elution Buffer A is stable for 30 days, Elution Buffer B is stable for 90 days, and Wash/Diluent Solution is stable for 60 days, when stored at 15–30 °C. The reconstituted Calibrators are stable for 24 hours when stored capped at 2–8 °C. The labeling indicates “Do not use the reconstituted Calibrators after 24 hours.”

Sample Stability:

A study was conducted to show the stability of frozen samples collected in K₂ and K₃EDTA. Samples with concentration values spanning 3.50% to 20.60% HbA1c were collected in K₂-EDTA tubes, K₃-EDTA tubes and the aliquots of the whole blood samples were placed at -70°C for four months. Study protocols were reviewed and found to be acceptable. The data supports the use of K₂-EDTA tubes and K₃-EDTA tubes with frozen whole blood aliquoted and stored at -70°C for up to for 4 months when using the VARIANT II TURBO HbA1c Kit 2.0.

d. Detection limit:

Not Applicable

e. Analytical specificity:

i.) Endogenous Interference:

Studies were performed to assess common or known substances that could interfere with the VARIANT II TURBO HbA1c Kit-2.0. Whole blood samples with HbA1c values of ~6.5% HbA1c and ~8% HbA1c were analyzed by spiking the interfering substance into each of the two whole blood samples and then preparing serial dilutions to achieve 10 concentrations. Ten replicates of each of the ten varying concentrations were analyzed and compared to the reference sample (sample containing no interferent). Significant interference was defined by the sponsor as % recovery ≥ +/-7% of the expected 100% recovery.

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The following substances showed no significant interference at the concentrations described below:

Endogenous Interference Study Results

| Endogenous Substance | Concentration |
| --- | --- |
| | Conventional Units |
| Lipemia (Intralipid) | 6000 mg/dL |
| Conjugated bilirubin | 60 mg/dL |
| Unconjugated bilirubin | 60 mg/dL |
| Glucose | 2000 mg/dL |
| Rheumatoid factor | 750 IU/mL |
| Total protein | 21 g/dL |

ii.) Drug Interference:

A Drug Interference study was performed based per CLSI EP07-A2, Interference Testing in Clinical Chemistry. Two EDTA whole blood sample pools were evaluated using a whole blood sample with a concentration ~6.5% HbA1c and whole blood sample with a concentration of ~8.0% HbA1c. Test samples were prepared by spiking each drug at the interferent concentration shown in the Table below. Ten replicates of each drug prepared with the test and control samples were analyzed using the VARIANT™ II TURBO HbA1c Kit-2.0 on the VARIANT™ II TURBO Hemoglobin Testing System.

Significant interference was defined as a more than ± 7% change in %HbA1c value from the control. No significant interference was observed at therapeutic levels up to the stated concentrations in the table below.

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Drug Interference Study Results

| Potential Drug Interferent | Highest Level Tested showing no Significant Interference |
| --- | --- |
| | Conventional (US) units |
| Acetylcysteine | 166 mg/dL |
| Ampicillin-Na | 1000 mg/dL |
| Ascorbic acid | 300 mg/dL |
| Cefoxitin | 2500 mg/dL |
| Heparin | 5000 U/L |
| Levodopa | 20 mg/dL |
| Methyldopa | 20 mg/dL |
| Metronidazole | 200 mg/dL |
| Doxycyclin | 50 mg/dL |
| Acetylsalicylic acid | 1000 mg/dL |
| Rifampicin | 64 mg/L |
| Cyclosporine | 5 mg/L |
| Acetaminophen | 200 mg/L |
| Ibuprofen | 500 mg/L |
| Theophylline | 100 mg/L |
| Phenylbutazone | 400 mg/L |

iii.) Cross Reactivity with Hemoglobin Derivatives:

A Hemoglobin Derivatives Interference study was performed based on CLSI EP07-A2, Interference Testing in Clinical Chemistry. Potential interference from Acetylated hemoglobin (Hb), Carbamylated hemoglobin (Hb) and Labile HbA1c were evaluated using a whole blood sample with a concentration ~6.5% HbA1c and a whole blood sample with a concentration of ~8.0% HbA1c. The potentially interfering hemoglobin derivatives were spiked into the whole blood samples and each sample was analyzed using ten replicates each in the same analytical run on the VARIANT™ II TURBO Hemoglobin Testing System with the VARIANT™ II TURBO HbA1c Kit – 2.0.

Significant interference was defined as more than a ±7% change in HbA1c value from the control. The test result conclusions are as follows:

- Acetylated Hb- (up to 50 mg/dL) does not interfere with this assay.
- Carbamylated Hb - (up to 21 mg/dL) does not interfere with this assay.
- Labile A1c- (up to 1000 mg/dL) glucose does not interfere with this assay.

Results showed there was no cross reactivity with these substances at physiological levels.

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iv.) Hemoglobin Variant Interference:

A Hemoglobin Variant Interference study was performed using a total of 147 samples known to contain hemoglobin variants S, C, E, D, A2 and F. Testing of the samples was performed using the VARIANT™ II TURBO HbA1c Kit – 2.0 on the VARIANT™ II TURBO Hemoglobin Testing System and compared to results obtained by a reference method that has been demonstrated to be free from the hemoglobin interferent being tested. The following is a table of the samples that were measured.

Variant samples used in Hemoglobin Variant Interference Study

| Hemoglobin Variant | n | Range in % Abnormal Variant | Range in %HbA1c Concentration |
| --- | --- | --- | --- |
| HbC | 25 | 33.3 – 42.4 | 4.50 - 10.70 |
| HbD | 21 | 30.2 – 41.4 | 5.5 - 11.4 |
| HbE | 24 | 24.7 - 31.4 | 5.0 - 8.3 |
| HbS | 26 | 26.8 - 41.6 | 4.70 - 13.3 |
| HbA2 | 22 | 5.0 – 10.2 | 5.0 – 14.5 |
| HbF | 29 | 3.5 – 29.4 | 4.4 – 14.4 |

The following results were obtained and are represented as a mean measurement with the standard deviation in parenthesis for the samples near 6.5 % HbA1c and 8.0 % HbA1c.

Hemoglobin Variant Results Summary

| Hemoglobin Variant | Relative % Bias from Reference Method at Low and High Concentrations of HbA1c | |
| --- | --- | --- |
| | Relative % Bias (StDev) for HbA1c ~6.5% | Relative % Bias (StDev) for HbA1c ~8.0% |
| HbC | -0.3 (+/- 3.5) | -2.5 (+/- 2.5) |
| HbD | -1.1 (+/- 1.7) | -1.2 (+/- 1.0) |
| HbE | 0.7 (+/- 3.0) | 2.2 (+/- 1.4) |
| HbS | 1.9 (+/- 2.8) | 2.8 (+/- 1.8) |
| HbA2 | 1.4 (+/- 2.3) | 2.0 (+/-4.1) |
| HbF | -1.9 (+/- 3.1) | -0.1 (+/-2.1) |

Non-significant interference was defined at ±7% from the control. No significant interference was observed for HbC (≤42.4%), HbD (≤41.4%), HbE (≤31.4%), HbS (≤41.6%), HbA2 (≤10.2%) and HbF (≤29.4%) variants at the concentrations tested in this Study.

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f. Assay cut-off:

Not Applicable

# 2. Comparison studies:

a. Method comparison with predicate device:

A Method comparison study was performed per CLSI EP09-A2 IR, Method Comparison and Bias Estimation Using Patient Samples. 130 variant-free whole blood EDTA samples, including 10 spiked samples, ranging from $3.4\%$ to $20.0\%$ HbA1c were evaluated using the VARIANT™ II TURBO HbA1c Kit- 2.0 on the VARIANT™ II TURBO Hemoglobin Testing System. Samples were tested in singlicate over several days using one lot of reagents. The results were compared to testing performed at a NGSP Secondary Reference Laboratory using a previously cleared HPLC HbA1c assay method (Trinity Bio-Tech Ultra2). To support the diagnostic claim, the distribution of samples spanned around the clinical decision point as follows in the table below.

Distribution of samples

| Hemoglobin A1c level | n | % Samples tested |
| --- | --- | --- |
| ≤ 5% | 6 | 4.6 |
| 5 – 6% | 17 | 13.1 |
| 6 – 6.5% | 33 | 25.4 |
| 6.5 – 7% | 31 | 23.8 |
| 7 – 8% | 21 | 16.2 |
| 8 – 9% | 11 | 8.5 |
| > 9% | 11 | 8.5 |
| Total samples | 130 | 100 |

Deming (weighted) and Passing-Bablok regression analyses were performed for the VARIANT™ II TURBO HbA1c Kit - 2.0 versus the NGSP SRL reference method.

Summary of Method Comparison Results

| | y-Intercept | 95% CI | Slope | 95% CI |
| --- | --- | --- | --- | --- |
| Deming | -0.275 | -0.342 to -0.208 | 1.033 | 1.023 to 1.043 |
| Passing-Bablok | -0.331 | -0.419 to -0.255 | 1.041 | 1.029 to 1.054 |

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Scatter Plot using Deming Fit, %HbA1c, NGSP SRL vs. VARIANT II TURBO HbA1c Kit - 2.0

Scatter Plot using Passing &amp; Bablok Fit, %HbA1c, NGSP SRL vs. VARIANT II TURBO HbA1c Kit - 2.0

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The following biases between VARIANT™ II TURBO HbA1c Kit – 2.0 versus the Reference method were observed:

Bias Estimation

| % HbA1c – Decision Level | Bias | % Bias |
| --- | --- | --- |
| 5.0 | -0.106 | -2.11 |
| 6.5 | -0.057 | -0.87 |
| 8.0 | -0.008 | -0.09 |
| 12.0 | 0.123 | 1.03 |

Using the results of bias estimation (%Bias) in the method comparison study and precision estimates in the reproducibility study, Total Error (TE) at four concentrations: (5.0%, 6.5%, 8.0% and 12.0%) were calculated as follows: $\% \mathrm{TE} = |\% \mathrm{Bias}| + 1.96 * \mathrm{CV}^* (1 + \% \mathrm{Bias})$ . The results are presented in the Table below.

Total Error Estimation

| %A1c | % Bias | % CV | % TE |
| --- | --- | --- | --- |
| 5.0 | -2.11 | 1.6 | 5.2 |
| 6.5 | -0.87 | 1.3 | 3.4 |
| 8.0 | -0.09 | 1.3 | 2.6 |
| 12.0 | 1.03 | 1.1 | 3.2 |

# b. Matrix comparison:

A matrix study was performed to determine the suitability of $\mathrm{K}_2$ -EDTA and $\mathrm{K}_3$ -EDTA anticoagulants used with fresh whole blood for use in the VARIANT II TURBO HbA1c Kit - 2.0. An additional study was performed to determine the suitability of Capillary blood in Hemoglobin Capillary Collection System (HCCS) for use in the VARIANT II TURBO HbA1c Kit - 2.0. Specimens with concentration values spanning 3.5 to $20.6\%$ HbA1c were collected from a minimum of 44 paired samples collected from different donors in $\mathrm{K}_2$ -EDTA tubes, $\mathrm{K}_3$ -EDTA tubes, and in the Capillary blood in Hemoglobin Capillary Collection System (HCCS).

In this Matrix Comparison study, the following tube types under evaluation:

- $\mathrm{K}_{2}$ -EDTA tubes with fresh whole blood
- $\mathrm{K}_{3}$ -EDTA tubes with fresh whole blood
- Hemoglobin Capillary Collection System (HCCS) with fresh capillary blood

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The regression results are as follows:

| | Matrix Comparison Results | | | |
| --- | --- | --- | --- | --- |
| Collection Device and Sample Type | Linear Fit | | | |
| | Sample Range | Slope (95% CI) | Intercept (95% CI) | r² value |
| K3-EDTA versus K2-EDTA | 3.5 – 20.6 | 0.997 | 0.031 | 0.9995 |
| | | 0.991 to 1.004 | -0.023 to 0.084 | |
| Capillary blood in Hemoglobin Capillary Collection System (HCCS) versus K3-EDTA whole blood. | 3.5 – 20.6 | 0.992 | 0.057 | 0.9993 |
| | | 0.984 to 0.999 | -0.007 to 0.121 | |

The data support the use of the following blood collection tubes and sample types with the VARIANT II TURBO HbA1c Kit – 2.0:

- K3-EDTA tubes with fresh whole blood
- K2-EDTA tubes with fresh whole blood
- Hemoglobin Capillary Collection System (HCCS) with fresh capillary blood

3. Clinical studies:

a. Clinical Sensitivity:

Not applicable

b. Clinical specificity:

Not applicable

c. Other clinical supportive data (when a. and b. are not applicable):

Not applicable

4. Clinical cut-off:

Not applicable

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5. Expected values/Reference range:

Hemoglobin A1c Expected Values

| Hemoglobin A1c | | |
| --- | --- | --- |
| NGSP% | IFCC mmol/mol | |
| >6.5 | >48 | Diabetic^{1-3} |
| 5.7 — 6.4 | 39 — 47 | Pre-Diabetic^{1} |
| < 5.7 | < 39 | Non-Diabetic |

1. American Diabetes Association. Diagnosis and Classification of Diabetes Mellitus. Diabetes Care 2010, 33 (Suppl. 1), S62–S69.
2. International Expert Committee. Report on the Role of the A1c Assay in the Diagnosis of Diabetes. Diabetes Care 2009, 32 (7), 1327–1334.
3. World Health Organization. Use of Glycated Hemoglobin (HbA1c) in the Diagnosis of Diabetes Mellitus. http://www.who.int/diabetes/publications/diagnosis_diabetes2011/en/ (accessed July 2014).

N. Proposed Labeling:

The labeling is sufficient and it satisfies the requirements of 21 CFR Part 809.10.

O. Conclusion:

The submitted information in this premarket notification is complete and supports a substantial equivalence decision.

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