VARIANT II TURBO HBA1C KIT KIT-20 AND WASH/DILUENT SOLUTION SET, MODELS 270-2455, 270-2730

K090699 · Bio-Rad Laboratories, Inc. · LCP · Jul 27, 2009 · Hematology

Device Facts

• Record ID: K090699
• Device Name: VARIANT II TURBO HBA1C KIT KIT-20 AND WASH/DILUENT SOLUTION SET, MODELS 270-2455, 270-2730
• Applicant: Bio-Rad Laboratories, Inc.
• Product Code: LCP · Hematology
• Decision Date: Jul 27, 2009
• Decision: SESE
• Submission Type: Traditional
• Regulation: 21 CFR 864.7470
• Device Class: Class 2

Indications for Use

The Bio-Rad VARIANT™ II TURBO HbA₁c Kit -2.0 is intended for the percent determination of hemoglobin A₁c in human whole blood using ion-exchange high-performance liquid chromatography (HPLC). Bio-Rad VARIANT™ II TURBO HbA₁c kit is intended for Professional Use Only. Measurement of percent hemoglobin A₁c is effective in monitoring long-term glucose control in individuals with diabetes mellitus.

Device Story

The VARIANT II TURBO HbA1c Kit - 2.0 is an in vitro diagnostic assay for professional use in clinical laboratories. It utilizes cation-exchange high-performance liquid chromatography (HPLC) to separate and quantify hemoglobin A1c from human anticoagulated whole blood (EDTA). The system processes samples through an analytical cartridge using sodium perchlorate buffers; detection occurs at 415 nm. The device provides a quantitative percentage of HbA1c, which clinicians use to monitor long-term glycemic control in diabetic patients. The system is standardized to DCCT and IFCC reference methods and certified by the NGSP. It is designed for high-throughput testing, with the kit supporting 2500 tests per cartridge.

Clinical Evidence

Bench testing only. Precision evaluated per CLSI EP5-A2 (within-device CV 0.91-1.15%). Linearity confirmed per CLSI EP6-A (3.5-19.0% HbA₁c). Interference testing performed for bilirubin, lipids, EDTA, labile A₁c, carbamylated hemoglobin, and HbF. Method comparison against predicate (n=52) showed correlation coefficient of 0.994. No clinical studies performed.

Technological Characteristics

Cation-exchange HPLC; sodium perchlorate buffer system; 415 nm optical detection; linear range 3.5–19.0% HbA1c; standardized to DCCT/IFCC/NGSP; professional use; EDTA whole blood sample input.

Indications for Use

Indicated for monitoring long-term glucose control in individuals with diabetes mellitus via measurement of percent hemoglobin A1c in human whole blood.

Regulatory Classification

Identification

A glycosylated hemoglobin assay is a device used to measure the glycosylated hemoglobins (A1a , A1b , and A1c ) in a patient's blood by a column chromatographic procedure. Measurement of glycosylated hemoglobin is used to assess the level of control of a patient's diabetes and to determine the proper insulin dosage for a patient. Elevated levels of glycosylated hemoglobin indicate uncontrolled diabetes in a patient.

Predicate Devices

• Variant II Hemoglobin A₁c Program (k070452)

Submission Summary (Full Text)

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

A. 510(k) Number:
k090699

B. Purpose for Submission:
Modification to a marketed device

C. Measurand:
Hemoglobin A₁c (HbA₁c)

D. Type of Test:
Quantitative 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

G. Regulatory Information:
1. Regulation section:
21 CFR § 864.7470, Glycosylated Hemoglobin Assay
2. Classification:
Class II
3. Product code:
LCP, Assay, Glycosylated Hemoglobin
4. Panel:
Clinical Chemistry (75)

H. Intended Use:
1. Intended use(s):
The Bio-Rad VARIANT™ II TURBO HbA₁c Kit -2.0 is intended for the percent determination of hemoglobin A₁c in human whole blood using ion-exchange high-performance liquid chromatography (HPLC). Bio-Rad VARIANT™ II TURBO HbA₁c kit is intended for Professional Use Only.

Measurement of percent hemoglobin A₁c is effective in monitoring long-term glucose control in individuals with diabetes mellitus.

2. Indication(s) for use:
See intended use above.

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3. Special conditions for use statement(s):
For Prescription Use Only

4. Special instrument requirements:
For use with the Bio-Rad VARIANT II TURBO Hemoglobin Testing System

I. Device Description:
The VARIANT II TURBO Hemoglobin Testing System provides an integrated method for sample preparation, separation and the percent determination of HbA₁c in EDTA human whole blood. The VARIANT II TURBO Hemoglobin Testing System is a fully automated, high-throughput hemoglobin analyzer. It consists of two modules - the VARIANT II Chromatographic Station (VCS) and the VARIANT II Sampling Station (VSS). In addition, a personal computer is used to control the VARIANT II System using Clinical Data Management (CDM) software versions 3.6T or 4.0.

The proposed device contains an analytical cartridge, 5 prefilters, Elution Buffers A and B, Calibrator Level 1, Calibrator Level 2, Whole Blood Primer, sample vials and a CD-ROM with test parameters.

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.

J. Substantial Equivalence Information:

1. Predicate device name(s):
Variant II Hemoglobin A₁c Program, Bio-Rad Laboratories, Inc.

2. Predicate K number(s):
k070452

3. Comparison with predicate:

| Item | Device | Predicate (k070452) |
| --- | --- | --- |
| Similarities | | |
| Intended use | Intended for the percent determination of HbA₁c in human whole blood using ion-exchange HPLC.
Measurement of percent HbA₁c is effective in monitoring long-term glucose control in individuals with diabetes mellitus. | same |
| Assay principle | Cation exchange HPLC | same |
| Analytical | Resin formulation | same |

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| cartridge | | | | | | |
| --- | --- | --- | --- | --- | --- | --- |
| Sample type | Anticoagulated whole blood (EDTA) | | | same | | |
| Visible detection | 415 nm | | | same | | |
| Standardization | Traceable to the Diabetes Control and Complication Trial (DCCT) reference method and IFCC. Certified via the National Glycohemoglobin Standardization Program (NGSP) | | | same | | |
| Calibrators | 2 levels | | | same | | |
| Interference | Bilirubin: no interference up to 20 mg/dL Lipids: no interference up to 6000 mg/dL EDTA: no interference up to 11X EDTA | | | same | | |
| Differences | | | | | | |
| Elution buffers | Sodium perchlorate buffer | | | Tris-bis buffer | | |
| Calibration | Analytical cartridge must be calibrated after priming a new cartridge. | | | Analytical cartridge must be calibrated after priming a new cartridge, with each lot of buffers and every 30 days. | | |
| Kit size | 2500 tests | | | 1000 tests | | |
| Total area range | Total area range of 1.0 to 3.5 million μvolt•second | | | Total area range of 1.5 to 4.5 million μvolt•second | | |
| Peak window names | In addition to A1a, A1b, F, LA1c, A1c, P3, P4, Ao and C, the candidate device has a variant window that includes HbE, C and S. | | | In addition to A1a, A1b, F, LA1c, A1c, P3, P4, Ao and C, the predicate has ED and S windows. | | |
| Precision | | Normal Patient | Diabetic Patient | | Normal Patient | Diabetic Patient |
| | Mean (% HbA1c) | 5.6 | 11.4 | Mean (% HbA1c) | 5.5 | 8.8 |
| | Within run (% CV) | 0.78 | 0.39 | Within run (% CV) | 0.90 | 0.59 |
| | Within device (% CV) | 1.15 | 0.91 | Within device (% CV) | 1.60 | 1.38 |
| Linear range | 3.5 to 19.0% HbA1c | | | 3.1 to 18.5% HbA1c | | |
| Interference | Hemoglobin F: No interference up to 25% Hemoglobin variants: Two out of 7 hemoglobin AD-trait, 2 out of 11 hemoglobin AS-trait, 1 out of 12 hemoglobin AE-trait, and 3 out of 9 hemoglobin AC-trait patient samples at the clinically significant levels of 6% and 9% HbA1c exhibited differences of more than ±10% from values obtained using boronate affinity reference method. | | | Hemoglobin F: No interference up to 15% Hemoglobin variants: no interference. | | |

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

- Evaluation of Precision Performance of Quantitative Measurement Methods; Approved Guideline--Second Edition (CLSI EP5-A2)
- Evaluation of the Linearity of Quantitative Measurement Procedures: A Statistical Approach; Approved Guideline (CLSI EP6-A)
- Interference Testing in Clinical Chemistry; Approved Guideline--Second Edition (CLSI EP7-A2)

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- Draft Guidance Document for 510(k) Submission of Glycohemoglobin (Glycated or Glycosylated) Hemoglobin for IVDs.
- Guidance for Industry and FDA Staff; Replacement Reagent and Instrument Family Policy
- Format for Traditional and Abbreviated 510(k)s - Guidance for Industry and FDA Staff
- Guidance for Industry and FDA Staff - Use of Symbols on Labels and in Labeling of In Vitro Diagnostic Devices Intended for Professional Use

## L. Test Principle:

The candidate device is a well established method of measuring the level of $\mathrm{HbA_{1c}}$ in the red blood cell. The candidate device is based on chromatographic separation of $\mathrm{HbA_{1c}}$ 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 $\mathrm{A_{1c}}$ , hemoglobin F and carbamylated hemoglobin.

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

### 1. Analytical performance:

#### a. Precision/Reproducibility:

The precision of the proposed device was evaluated using a protocol based on the CLSI EP5-A2 guideline. Normal patient, normal control, diabetic patient and diabetic control samples were run in the precision study. The precision study was performed on 6 instruments in 3 laboratories over 10 days. Each site was provided with the same sample set and directed to perform 2 replicates of each sample on each of 2 runs/day (morning and evening) for 10 days. Each site conducted the study on 2 instruments. The position of the precision specimens in each run was randomized. The results were analyzed by nested Analysis of Variance (ANOVA) with the hierarchical levels of labs, instruments, days, and runs. Patient samples are EDTA human whole blood. The results are summarized below:

| | Normal patient | Diabetic patient |
| --- | --- | --- |
| Mean (% HbA1c) | 5.6 | 11.4 |
| Within-run (% CV) | 0.78 | 0.39 |
| Between-day (% CV) | 0.66 | 0.69 |
| Between-run (% CV) | 0.53 | 0.45 |
| Within-device (% CV) | 1.15 | 0.91 |
| Total precision (% CV) | 2.01 | 1.93 |

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| | Normal control | Diabetic control |
| --- | --- | --- |
| Mean (% HbA1c) | 5.6 | 10.0 |
| Within-run (% CV) | 1.09 | 0.62 |
| Between-day (% CV) | 0.59 | 0.62 |
| Between-run (% CV) | 0.00 | 0.33 |
| Within-device (% CV) | 1.24 | 0.94 |
| Total precision (% CV) | 2.08 | 2.10 |

b. Linearity/assay reportable range:

The study was performed following the CLSI EP6-A guideline. Linearity across the reportable range was performed using low (2.5% HbA₁c) and high (19.6% HbA₁c) EDTA whole blood patient samples. These were mixed together in varying ratios. The measured values were compared to the theoretical values based upon the dilution factor. 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. The measured results for each dilution were within a maximum difference of 0.24% in this range. The results are summarized below:

| % A1c (theoretical) | Predicted 1storder | Predicted 3rdorder | Difference |
| --- | --- | --- | --- |
| 2.55 | 2.82 | 2.58 | 0.24 |
| 5.04 | 5.26 | 5.34 | -0.08 |
| 7.10 | 7.28 | 7.45 | -0.17 |
| 9.52 | 9.66 | 9.79 | -0.13 |
| 11.08 | 11.20 | 11.26 | -0.06 |
| 13.63 | 13.70 | 13.64 | 0.06 |
| 15.56 | 15.59 | 15.47 | 0.12 |
| 17.61 | 17.61 | 17.52 | 0.09 |
| 19.60 | 19.56 | 19.63 | -0.07 |

The reportable range of the device is 3.5 to 19.0% HbA₁c.

c. Traceability, Stability, Expected values (controls, calibrators, or methods): Calibrators were previously cleared in k070452. The recommended control materials were cleared in k070546 and k052838.

The proposed device has been certified by the National Glycohemoglobin Standardization Program (NGSP) as having documented traceability to the Diabetes Control and Complications Trial reference method.

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d. Detection limit:

The reportable range is 3.5 to 19.0 % HbA₁c (see linearity section above).

e. Analytical specificity:

Icterus (bilirubin) and lipemia (triglyceride) interference: Two EDTA whole blood patient samples representing normal and diabetic HbA₁c samples were split into aliquots and treated with increasing concentrations of triglycerides and bilirubin. The treated and untreated samples were run in duplicate on a VARIANT II TURBO Hemoglobin Testing System and the differences in % HbA₁c between the samples were analyzed. Bilirubin up to 20 mg/dL and triglycerides up to 6000 mg/dL had no significant effect on HbA₁c determination.

EDTA interference: In order to determine the effect of high concentrations of EDTA that can occur in cases of "short draws", 2 whole blood patient samples representing normal and diabetic HbA₁c samples were split into aliquots and treated with 11 times (11X) the expected EDTA concentration in samples. The treated and untreated samples were run in duplicate on a VARIANT II TURBO Hemoglobin Testing System and the differences in % HbA₁c between the samples were analyzed. EDTA, up to 11X had no significant effect on HbA₁c determination.

Labile A₁c (LA₁c) interference: Two EDTA whole blood patient samples representing normal and diabetic HbA₁c samples were split into aliquots and treated with increasing concentrations of a glucose solution (up to 1000 mg/dL) and incubated for 3 hours at 37 °C to facilitate the formation of LA₁c. The treated samples and untreated control samples were run in duplicate on two VARIANT II TURBO Hemoglobin Testing Systems and the differences in % HbA₁c between the samples were analyzed. LA₁c up to 6% had no significant effect on HbA₁c determination.

Carbamylated hemoglobin interference: A series of EDTA whole blood patient samples representing normal and diabetic HbA₁c were prepared with varying concentrations of a high carbamylated hemoglobin solution (prepared by treating the red blood cells of a normal patient sample and a diabetic patient sample with 2.6 mM potassium cyanate at 37 °C for 45 minutes until the carbamylated hemoglobin level increased to 6%). The carbamylated-containing samples and the control samples were run in duplicate on a VARIANT II TURBO Hemoglobin Testing System and the differences in % HbA₁c between the samples were analyzed. Carbamylated hemoglobin up to 4% had no significant effect on HbA₁c determination.

Hemoglobin F (HbF) Interference: A series of EDTA whole blood patient samples representing normal and diabetic HbA₁c were prepared with varying concentrations of a high HbF (>25 %) solution. The treated samples and untreated control samples were run in duplicate on two VARIANT II TURBO

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Hemoglobin Testing Systems and the differences in % HbA₁c between the samples were analyzed. HbF up to 25% had no significant effect on HbA₁c determination.

Hemoglobin Variant Interferences: Hemoglobin AD, AE, AS and AC-trait patient samples (20 of each) with HbA₁c values distributed across the non-diabetic and diabetic range were analyzed by the proposed device in replicates of four and compared to values obtained by a NGSP secondary Reference Laboratory using a boronate affinity method. Two out of 7 hemoglobin AD-trait, 2 out of 11 hemoglobin AS-trait, 1 out of 11 hemoglobin AE-trait and 3 out of 9 hemoglobin AC-trait patient samples within the clinically significant range of 6% and 9% HbA₁c tested exhibited differences of more than ± 10% from values obtained using the reference method.

The sponsor includes the following statements in the product insert:

- Icterus, as indicated by bilirubin concentrations up to 20 mg/dL, does not interfere with the assay.
- Lipemia, as indicated by testing a normal and a diabetic sample spiked with a lipemic clinical serum sample to achieve triglyceride concentrations up to 6000 mg/dL, does not interfere with the assay.
- Carbamylated hemoglobin concentrations up to 4% do no interfere with the assay.
- Hemoglobin F concentrations up to 25% do not interfere with the assay. In cases where hemoglobin F concentrations are greater than 25%, the % A₁c should not be reported.
- In a study to assess interference form hemoglobin variants, 2 out of 7 hemoglobin AD-trait, 2 out of 11 hemoglobin AS-trait, 1 out of 11 hemoglobin AE-trait and 3 out of 9 hemoglobin AC-trait patient samples within the clinically significant range of 6% and 9% HbA₁c tested exhibited differences of more than ± 10% from values obtained using the reference method. Laboratories should take this into consideration when evaluating results from patients with a hemoglobin variant trait.

f. Assay cut-off:

Not applicable.

2. Comparison studies:

a. Method comparison with predicate device:

To demonstrate accuracy across the measuring range of 3.5 to 19% HbA₁c, the proposed device was compared to the predicate using 40 EDTA whole blood patient samples and 12 spiked EDTA whole blood samples. The samples were run in singlicate on the proposed device and on the predicate device. A linear regression analysis of the results calculated a slope of 0.9621, an intercept of 0.4443, and a correlation coefficient of 0.994.

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A second study was performed to evaluate the proposed device compared to the previous VARIANT II TURBO Hemoglobin A1c method (cleared in k040872 and the software was modified and cleared in k063400). A comparison of 42 EDTA whole blood patient samples with values distributed from 4.8 to 12.5 % HbA₁c (on the proposed device) were run in singlicate on both systems. A linear regression analysis of these results calculated a slope of 1.0957, an intercept of 0.7375, and a correlation coefficient of 0.9952.

b. Matrix comparison: Not applicable.

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.

5. Expected values/Reference range: Hemoglobin A1c Expected Value Range was cited from the American Diabetes Association. Standards of Medical Care for Patients with Diabetes Mellitus. Diabetes Care 2001, 24 (Suppl. 1), 33-34.

| Hemoglobin A1c (%) | Degree of Glucose Control |
| --- | --- |
| > 8 | Action Suggested |
| < 7 | Goal |
| < 6 | Non-Diabetic Level |

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.