The D-100™ HbA1c test 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 D-100 Hemoglobin Testing System.

Hemoglobin A1c measurements are used as an aid in diagnosis of diabetes mellitus, as an aid to identify patients who may be at risk for developing diabetes mellitus, and for the monitoring of long-term blood glucose control in individuals with diabetes mellitus.

The D-100TM HbA1c test is intended for Professional Use Only.

Calibrators:

The D-100™ HbA1c Calibrator Pack is for the calibration of the D-100 Hemoglobin Testing System used for the quantitative determination of hemoglobin A1c(HbA1c) in human whole blood.

Device Description

The Bio-Rad D-100™ HbA1c utilizes principles of ion-exchange high-performance liquid chromatography (HPLC). A high-pressure pumping system delivers a buffer solution to an analytical cartridge and detector. Whole blood samples undergo an automatic hemolysis and dilution process before being introduced into the analytical flow path. Prediluted samples are identified based upon the use of a microvial adapter in the sample rack, and the automatic dilution step is omitted.

A programmed buffer gradient of increasing ionic strength delivers the sample to the analytical cartridge where the hemoglobin species are separated based upon their ionic interactions with the cartridge material and the buffer gradient. The separated hemoglobin species then pass through the flow cell where changes in the absorbance are measured at 415 nm and recorded as a digital chromatogram.

The software performs an analysis of the hemoglobin peaks in the chromatogram, recording information including retention time, peak area, and relative are percent. Any peaks that are identified as the target analyte(s) are calibrated before generating a sample report and chromatogram for each sample. The software includes an optional feature (Advisor) that compares the sample report against a set of rules that are programmed to take user-specified actions.

The D-100™ HbA1c test is designed to be used on the D-100™ Hemoglobin Testing System.

AI/ML Overview

This document describes the D-100™ HbA1c test and D-100™ HbA1c Calibrator Pack, intended for the quantitative determination of hemoglobin A1c (HbA1c) in human whole blood using ion-exchange high-performance liquid chromatography (HPLC) on the D-100™ Hemoglobin Testing System. The test is used as an aid in diagnosing diabetes mellitus, identifying individuals at risk for diabetes, and monitoring long-term blood glucose control in diabetic patients. The calibrators are used for the calibration of the system.

1. Table of Acceptance Criteria and Reported Device Performance

The acceptance criteria are implied through the study designs, particularly the "significant interference" threshold for analytical specificity and the performance demonstrated in linearity and precision studies. For the purpose of this summary, the relevant performance metrics are extracted and presented.

Performance Metric	Acceptance Criteria (Implied)	Reported Device Performance
Precision (NGSP %CV Total Precision)	Not explicitly stated, but results within reasonable analytical variation for diagnostic assays.	< 1.8% for all samples (NGSP%) across all instruments, with combined total precision from 0.9% to 1.8%.
Precision (IFCC mmol/mol %CV Total Precision)	Not explicitly stated.	< 3.4% for all samples (IFCC mmol/mol) across all instruments, with combined total precision from 1.1% to 3.1%.
Linearity (NGSP % HbA1c)	Maximum measured difference of ±0.09% between 1st and 2nd order results (as shown in Table 11).	Demonstrated linearity from 3.5 - 20.0% HbA1c, with a maximum measured difference of ±0.09% between predicted 1st and 2nd order results.
Linearity (IFCC mmol/mol HbA1c)	Maximum measured difference of ±0.9% (or ±0.94 mmol/mol) between 1st and 2nd order results (as shown in Table 12).	Demonstrated linearity from 15 – 195 mmol/mol, with a maximum measured difference of ±0.94 mmol/mol.
Method Comparison (Deming Regression Slope for NGSP %)	Close to 1.0 (indicating agreement with reference).	0.9867 (95% CI: 0.9736 – 0.9999)
Method Comparison (Deming Regression y-Intercept for NGSP %)	Close to 0.0 (indicating agreement with reference).	0.0223 (95% CI: -0.0684 – 0.1131)
Method Comparison (Passing-Bablok Regression Slope for NGSP %)	Close to 1.0.	0.9909 (95% CI: 0.9789 – 1.0026)
Method Comparison (Passing-Bablok Regression y-Intercept for NGSP %)	Close to 0.0.	-0.0091 (95% CI: -0.0803 – 0.0763)
Total Error (TE)	Not explicitly stated, but calculated values like 4.2% at 5.0% A1c are presented.	Calculated Total Error (TE) values ranging from 3.6% to 4.2% across different HbA1c decision levels.
Analytical Specificity (Endogenous Interference)	< ±7% change in %HbA1c value from control.	No significant interference observed for Lipemia (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).
Analytical Specificity (Drug Interference)	< ±7% change in %HbA1c value from control.	No significant interference observed at therapeutic levels up to specified concentrations for 15 common drugs (e.g., Acetylcysteine, Ampicillin-Na, Ascorbic acid, Cefoxitin, Heparin, Levodopa, Methyldopa, Metronidazole, Doxycyclin, Acetylsalicylic acid, Rifampicin, Cyclosporine, Acetaminophen, Ibuprofen, Theophylline, Phenylbutazone).
Analytical Specificity (Hemoglobin Derivatives Cross-Reactivity)	< ±7% change in HbA1c value from control.	No interference from Acetylated Hb (up to 50 mg/dL), Carbamylated Hb (up to 5%), and Labile A1c (up to 1200 mg/dL of glucose).
Analytical Specificity (Hemoglobin Variants)	Not explicitly stated as a percentage bias threshold, but performance is compared against an NGSP reference method.	Relative %Bias from -0.6 to -4.7 for HbS, HbC, HbD, HbE, HbA2, and HbF variants at ~6.5% HbA1c, and from -1.3 to -4.4 at ~9.0% HbA1c. The study indicates the device is free from hemoglobin interferent from a comparable method.

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

Precision/Reproducibility:
- Sample Size: Four EDTA whole blood samples at target HbA1c concentrations (~5%, ~6.5%, ~8%, ~12%) and five quality control materials.
- Data Provenance: Not explicitly stated, but typically these samples would be from a clinical setting. Retrospective or prospective is not specified. The study was conducted at "two different sites."
Linearity:
- Sample Size: Low (3.5% HbA1c) and high (20% HbA1c) EDTA whole blood patient samples, mixed in varying ratios to create 11 sample pools.
- Data Provenance: Not explicitly stated, but these are patient samples. Retrospective or prospective is not specified.
Method Comparison:
- Sample Size: 129 variant-free whole blood EDTA samples.
- Data Provenance: Patient samples. Retrospective or prospective is not specified.
Analytical Specificity (Endogenous Interference):
- Sample Size: Two EDTA whole blood sample pools (one at ~6.5% HbA1c and one at ~8.0% HbA1c) for each interferent, with 10 replicates per test and control sample.
- Data Provenance: Whole blood samples. Serum samples were used for Rheumatoid factor, lipemia, and total protein (with known high concentrations), then mixed with whole blood. Retrospective or prospective is not specified.
Analytical Specificity (Drug Interference):
- Sample Size: Two EDTA whole blood sample pools (one at ~6.5% HbA1c and one at ~8.0% HbA1c) for each drug, with 10 replicates per test and control sample.
- Data Provenance: Whole blood samples. Retrospective or prospective is not specified.
Analytical Specificity (Hemoglobin Derivatives Cross-Reactivity):
- Sample Size: Low (~6.5% HbA1c) and high (~8.0% HbA1c) EDTA whole blood samples for each derivative, with 10 replicates for each sample pool.
- Data Provenance: Whole blood EDTA samples. Retrospective or prospective is not specified.
Hemoglobin Variant Study:
- Sample Size: 20 HbS, 20 HbC, 20 HbD, 20 HbE, 25 HbA2, and 30 HbF specific variant samples (total 135 samples). Two whole blood EDTA patient samples (one ~6.5% HbA1c, one ~8% HbA1c) with the appropriate variants tested in duplicate.
- Data Provenance: Patient samples known to contain specific hemoglobin variants. Retrospective or prospective is not specified.

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

For this in vitro diagnostic device, human experts are not directly used to establish the ground truth for the test set in the way they might be for image-based diagnostic AI. Instead, the ground truth is established through:

Reference Methods:
- Precision/Reproducibility: No separate "ground truth" establishment by experts listed, as it measures the device's inherent variability.
- Linearity: No separate "ground truth" establishment by experts listed; it assesses the device's response across a range compared to theoretical values.
- Method Comparison: The reference method was a "secondary NGSP SRL reference laboratory using a cleared HPLC-based HbA1c assay," implying highly controlled and standardized laboratory procedures.
- Analytical Specificity & Hemoglobin Variant Study: Comparison to "control" samples or an "NGSP reference method that has been demonstrated to be free from the hemoglobin interferent."

The "experts" in this context are the highly trained laboratory professionals and the robust, standardized analytical methods used in the reference laboratories (e.g., NGSP SRL reference laboratory). Specific number or individual qualifications of these professionals beyond "NGSP SRL reference laboratory" are not detailed in this summary.

4. Adjudication Method for the Test Set

Not applicable in the typical sense of expert adjudication (e.g., 2+1, 3+1 for imaging studies). The comparison is against established laboratory reference methods and defined control samples, which serve as the gold standard. Discrepancies would be resolved through re-testing or investigation per standard laboratory quality control protocols, not through a formal expert adjudication panel.

5. If a Multi-Reader Multi-Case (MRMC) Comparative Effectiveness Study was done, If so, what was the effect size of how much human readers improve with AI vs without AI assistance

Not applicable. This is an In Vitro Diagnostic (IVD) device, specifically an automated chemical analyzer (HPLC) for quantitative measurement of HbA1c. There are no "human readers" or "AI assistance" in the context of interpreting images or complex data as would be found in traditional MRMC studies for AI-powered diagnostics. The device completely automates the measurement.

6. If a Standalone (i.e., algorithm only without human-in-the-loop performance) was done

Yes, the studies presented are all standalone. The D-100™ HbA1c test and its associated system perform the quantitative determination of HbA1c without human intervention during the measurement process itself, beyond initial sample loading and system operation. The performance metrics (precision, linearity, method comparison, analytical specificity) are all measures of the device's inherent analytical capability.

7. The Type of Ground Truth Used

The ground truth used is primarily:

Reference Method / Standardized Assays: For method comparison, the device's results were compared to "testing performed at a secondary NGSP SRL reference laboratory using a cleared HPLC-based HbA1c assay." For the Hemoglobin Variant study, it was compared to an "NGSP reference method that has been demonstrated to be free from the hemoglobin interferent."
Known Concentrations/Prepared Samples: For linearity, known ratios of low and high concentration samples were used. For analytical specificity studies, interferents were prepared at known concentrations and spiked into samples.
Certified Materials: The calibrators are traceable to the International Federation of Clinical Chemistry (IFCC) reference method and are NGSP certified.

8. The Sample Size for the Training Set

Not applicable in the context of this device. The D-100™ HbA1c system is an HPLC-based analytical instrument. It is not an "algorithm" in the sense of a machine learning or AI model that requires a training set for model development. Its "training" involves calibration with certified reference materials (the Calibrator Pack), which are part of the overall analytical system.

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

Not applicable as there is no "training set" for an AI algorithm in the traditional sense. The calibration process (which could be conceptually analogous to "training" for an analytical instrument) uses the D-100™ HbA1c Calibrator Pack. The ground truth for these calibrators is established by:

Traceability to IFCC Reference Method: "Each Calibrator Pack contains Calibrator values which have been value assigned using secondary calibrators that are traceable to the International federation of Clinical Chemistry and Laboratory Medicine (IFCC) reference method."
NGSP Certification: The assay is NGSP certified, meaning its results correlate to the Diabetes Control and Complications Trial (DCCT) reference method.

Summary

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Food and Drug Administration 10903 New Hampshire Avenue Document Control Center - WO66-G609 Silver Spring, MD 20993-0002

BIO-RAD LABORATORIES, INC. JACKIE BUCKLEY REGULATORY AFFAIRS REP IV 4000 ALFRED NOBEL DR. HERCULES CA 94547

December 9, 2015

Re: K151321 Trade/Device Name: D-100™ HbA1c D-100™ HbA1c Calibrator Pack Regulation Number: 21 CFR 862.1373 Regulation Name: Glycosylated hemoglobin assay Regulatory Class: II Product Code: PDJ, LCP, JIT Dated: November 06, 2015 Received: November 09, 2015

Dear Jackie Buckley:

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. 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 Parts 801 and 809); medical device reporting (reporting of medical device-related adverse events) (21 CFR 803); good manufacturing practice requirements as set forth in the quality systems (OS) regulation (21 CFR Part 820); and if applicable, the electronic product radiation control provisions (Sections 531-542 of the Act); 21 CFR 1000-1050.

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If you desire specific advice for your device on our labeling regulations (21 CFR Parts 801 and 809), please contact the Division of Industry and Consumer Education at its toll-free number (800) 638 2041 or (301) 796-7100 or at its Internet address

http://www.fda.gov/MedicalDevices/ResourcesforYou/Industry/default.htm. 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

http://www.fda.gov/MedicalDevices/Safety/ReportaProblem/default.htm for the CDRH's Office of Surveillance and Biometrics/Division of Postmarket Surveillance.

You may obtain other general information on your responsibilities under the Act from the Division of Industry and Consumer Education at its toll-free number (800) 638-2041 or (301) 796-7100 or at its Internet address

http://www.fda.gov/MedicalDevices/ResourcesforYou/Industry/default.htm.

Sincerely yours,

Katherine Serrano -S

For: Courtney H. Lias, Ph.D. Director Division of Chemistry and Toxicology Devices Office of In Vitro Diagnostics and Radiological Health Center for Devices and Radiological Health

Enclosure

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Indications for Use

510(k) Number (if known) K151321

Device Name D-100™ HbA1c D-100™ HbA1c Calibrator Pack

Indications for Use (Describe)

The D-100TM HbA1c test is intended for Professional Use Only.

Calibrators:

The D-100™ HbA1c Calibrator Pack is for the calibration of the D-100 Hemoglobin Testing System used for the quantitative determination of hemoglobin A1c(HbA1c) in human whole blood.

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

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510(k) Summary (Summary of Safety and Effectiveness)

This Summary of 510(k) Safety and Effectiveness is being submitted in accordance with the requirements of SMDA 1990 and 21 CFR 807.92.

The assigned 510(k) number is: K151321

Date Summary prepared: Nov. 4, 2015

1. Applicant Name:

Bio-Rad Laboratories, Inc. Clinical Diagnostics Group 4000 Alfred Nobel Drive Hercules, California 94547

2. Contact Person(s):

Jackie Buckley, Regulatory Affairs Representative IV Telephone Number: (510) 741-5309 FAX: (510) 741-6471 E-Mail: jackie buckley@bio-rad.com

Alfred Evans, RA/QA Director Telephone Number: (510) 741-4579 FAX: (510) 741-6471 E-Mail:al_evans@bio-rad.com

3. Device Name/Trade Name:

Reagents:

Trade Name: D-100™ HbA1c Classification Name: Assay, Glycosylated Hemoglobin Common Name: HbA1c Product Code: PDJ, LCP C.F.R Section: 21 CFR 862.1373 Device classification: Class II Panel Classification: Chemistry

Calibrators:

Trade Name: D-100™ HbA1c Calibrator Pack Classification Name: Calibrator, Secondary Common Name: Calibrator Product Code: JIT C.F.R Section: 21 CFR 862.1150 Device classification: Class II Panel Classification: Clinical Chemistry

4. Predicate Device:

Predicate DeviceName	Predicate Device510(k) Number
VARIANT II TURBO HbA1c Kit -2.0	K142448
VARIANT II Hemoglobin A1c Calibrators	K070452

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5. Description of the Device:

The D-100™ HbA1c test is designed to be used on the D-100™ Hemoglobin Testing System.

Reagents:

The D-100™ HbA1c reagents contain the following components:

Description

D-100 ™ HbAic Analytical Cartridge/Calibrator Pack. One pack consisting of:

. Cation exchange cartridge. 10,000 tests each
. Calibrator Pack: 1 vial of Conditioner, 1 vial of Calibrator Level 1, and 1 vial of Calibrator Level 2. The vials contain Ivophilized human whole blood with glycine and trehalose as preservatives.

D-100 TM Prefilters. 2000 tests each. Package of 5.

D-100 ™ Cleaning Tube. One microvial containing 1.5 mL of a liquid cleaning solution. Single use.

D-100 ™ Sample Diluent. Each bottle contains 1 L of deionized water with <0.05% sodium azide as a preservative.

D-100 ™ HbAçe Elution Buffer A. Each bottle contains 2600 mL of a succinate/sodium perchlorate buffer. Contains <0.05% sodium azide as a preservative.

D-100 ™ HbAçe Elution Buffer B. Each bottle contains 1400 mL of a succinate/sodium perchlorate buffer. Contains<0.05% sodium azide as a preservative.

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D-100 ™ Wash Solution. Each bottle contains 3300 mL of deionized water with <0.05% sodium azide as a preservative.

Calibrator:

Each Calibrator Pack contains Calibrator values which have been value assigned using secondary calibrators that are traceable to the International federation of Clinical Chemistry and Laboratory Medicine (IFCC) reference method.

Description

D-100 "" HbAg Calibrator Pack. One pack consisting of 1 vial of Conditioner, 1 vial of Calibrator Level 1, and 1 vial of Calibrator Level 2. The vials contain lyophilized human whole blood with qlycine and trehalose as preservatives.

6. Indications for Use:

The D-100™ HbA1c test is intended for the quantitative determination of hemoglobin A1c (IFCC mmol/mol and NGSP %) in human whole blood using ion-exchange highperformance liquid chromatography (HPLC) on the D-100™ Hemoglobin Testing System.

Hemoglobin A1c measurements are used as an aid in diagnosis of diabetes, as an aid to identify patients who may be at risk for developing diabetes mellitus, and for the monitoring of long-term blood glucose control in individuals with diabetes mellitus.

The Bio-Rad D-100™ HbA1c test is intended for Professional Use Only.

Calibrators

The D-100 HbA1c Calibrator Pack is for the calibration of the D-100 Hemoglobin Testing System used for the quantitative determination of hemoglobin A1c (HbA1c) in human whole blood.

7. Substantial Equivalence Information:

Predicate Device Information:

Predicate DeviceName	Predicate Device510(k) Number
VARIANT II TURBO HbA1c Kit -2.0	K142448
VARIANT II Hemoglobin A1c Calibrators	K070452

The comparison of the technological characterizes of the D-100 HbA1c assay (candidate assay) utilizes principles of ion-exchange high-performance liquid chromatography (HPLC) similar to the same technology of the VARIANT II TURBO HbA1c Kit - 2.0 (predicate device).

Tables 1 and 2 provide the similarities and differences between the candidate assay and the predicate assay.

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ReagentSimilarities and Differences
Features	Candidate Device:D-100™ HbA1c(K)151321	Predicate Device:VARIANT™ II TURBO HbA1cKit – 2.0(K)142448
Intended Use	Same	Intended for thequantitative determination ofhemoglobin A1c (IFCC mmol/moland NGSP %)
Platform	D-100™ Hemoglobin TestingSystem	VARIANT™TURBO HemoglobinTesting System andVARIANT™TURBO LinkHemoglobin Testing System
MeasuringInterval	3.5 to 20% (NSGP)15 – 195 mmol/molHbA1c (IFCC)	3.4 to 20.6 % (NSGP)14 – 203 mmol/molHbA1c (IFCC)
Specimen Type	Same	Human Whole blood
Assay Principle	Same	Ion exchange HPLC
Matrices	K2-EDTA, K3-EDTAPotassium Oxalate/SodiumFluoride, Sodium Citrate,Sodium Heparin, LithiumHeparin	K2-EDTA, K3-EDTAHemoglobin Capillary Collection Kit
Standardization	Same	Traceable to the DiabetesControl and Complications Trial(DCCT) reference method andIFCC. Certified via the NationalGlycohemoglobin StandardizationProgram (NGSP)

Table 1: Reagent Similarities and Differences

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CalibratorSimilarities and Differences
Features	Candidate Device:D-100™ HbA1cCalibrator Pack(K)151321	Predicate Device:VARIANT™ II HemoglobinA1c Calibrators(K) 070452
Intended Use	Same	Intended for thequantitative determination ofhemoglobin A1c in Human WholeBlood
Levels	Same	Levels 1 & 2Calibration is performed once atthe beginning of a new cartridge.
Standardization/Traceability	Same	Each lot of calibrators is valueassigned and values are reportedin both NGSP and IFCC units.

Table 2: Calibrator Similarities and Differences

8. Summary of Nonclinical Performance Data:

a. Precision/Reproducibility:

The precision of the D-100™ HbA1c test 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 utilized in the study. In addition, five quality control materials were also tested. Precision was evaluated using three reagent lots, three D-100™ I Hemoglobin Testing Systems at two different sites. The samples were run in duplicate in 2 runs per day for 20 day. NGSP results are shown in Tables 3-6. IFCC results are shown in Tables 7-10.

Variation Source
	Instrument ID: SM93
	Control 1	Control 2	Patient 1	Patient 2	Patient 3	Patient 4	QC 1	QC 2	QC 3
ConcentrationHbA1c (NGSP%)	5.5%	9.4%	5.2%	6.7%	8.1%	12.0%	5.3%	9.9%	14.8%
Repeatability	0.7%	0.7%	0.7%	0.7%	0.7%	0.6%	0.8%	0.7%	0.7%
Between-Run	0.0%	0.1%	0.0%	0.0%	0.0%	0.0%	0.0%	0.0%	0.0%
Between-Day	0.6%	0.2%	0.3%	0.2%	0.1%	0.3%	0.3%	0.1%	0.2%
Between-Lot	1.2%	0.8%	1.3%	1.1%	1.0%	0.6%	1.4%	0.8%	0.6%
Total Precision	1.5%	1.1%	1.5%	1.3%	1.2%	0.9%	1.6%	1.0%	0.9%

Table 3: Instrument 1 (% CV by Sample (NGSP)

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Variation Source	Instrument ID: SM94
	Control 1	Control 2	Patient 1	Patient 2	Patient 3	Patient 4	QC 1	QC 2	QC 3
ConcentrationHbA1c (NGSP%)	5.5%	9.4%	5.2%	6.6%	8.1%	12.0%	5.3%	10.0%	14.8%
Repeatability	1.1%	0.9%	0.8%	0.9%	1.0%	0.9%	1.0%	1.0%	1.0%
Between-Run	0.0%	0.3%	0.0%	0.0%	0.2%	0.0%	0.0%	0.1%	0.1%
Between-Day	0.6%	0.2%	0.5%	0.5%	0.3%	0.3%	0.2%	0.4%	0.3%
Between-Lot	1.2%	0.2%	1.5%	0.6%	0.0%	0.3%	1.5%	0.1%	0.5%
Total Precision	1.7%	1.0%	1.8%	1.2%	1.0%	1.0%	1.8%	1.1%	1.1%

Table 4: Instrument 2 (% CV by Sample (NGSP)

Table 5: Instrument 3 (% CV by Sample (NGSP))

	Instrument ID: SM98
Variation Source	Control 1	Control 2	Patient 1	Patient 2	Patient 3	Patient 4	QC 1	QC 2	QC 3
ConcentrationHbA1c (NGSP%)	5.4%	9.4%	5.1%	6.6%	8.1%	12.0%	5.3%	9.9%	14.7%
Repeatability	1.0%	1.0%	1.0%	1.1%	0.9%	0.9%	1.0%	0.9%	0.8%
Between-Run	0.0%	0.0%	0.0%	0.0%	0.0%	0.0%	0.0%	0.0%	0.0%
Between-Day	0.6%	0.5%	0.4%	0.5%	0.5%	0.4%	0.4%	0.4%	0.4%
Between-Lot	1.6%	1.0%	1.5%	1.5%	1.2%	1.1%	1.7%	1.0%	1.1%
Total Precision	1.9%	1.4%	1.9%	2.0%	1.6%	1.5%	2.0%	1.4%	1.4%

Table 6: Instruments Combined (% CV by Sample (NGSP))

	All Instruments
Variation Source	Control1	Control2	Patient1	Patient2	Patient3	Patient4	QC 1	QC 2	QC 3
ConcentrationHbA1c (NGSP %)	5.5%	9.4%	5.2%	6.6%	8.1%	12.0%	5.3%	9.9%	14.8%
Repeatability	0.9%	0.9%	0.9%	0.9%	0.9%	0.8%	0.9%	0.9%	0.8%
Between-Run	0.0%	0.0%	0.0%	0.0%	0.1%	0.0%	0.0%	0.0%	0.0%
Between-Day	0.6%	0.3%	0.4%	0.4%	0.4%	0.3%	0.3%	0.3%	0.3%
Between-Instrument	0.0%	0.0%	0.0%	0.0%	0.0%	0.0%	0.0%	0.0%	0.0%
Between-Lot	1.4%	0.7%	1.5%	1.1%	0.9%	0.7%	1.5%	0.7%	0.8%
Total Precision	1.7%	1.2%	1.7%	1.5%	1.3%	1.2%	1.8%	1.2%	1.2%

Table 7: Instrument 1 (% CV by Sample (IFCC Units- mmol/mol))

Variation Source	Control1	Control2	Patient1	Patient2	Patient3	Patient4	QC 1	QC 2	QC 3
Instrument ID:SM93
ConcentrationHbA1c (IFCC)	36.5	79.3	33.2	49.4	65.4	108.1	34.5	85.0	137.7
Repeatability	1.2%	0.9%	1.2%	1.0%	0.9%	0.8%	1.3%	0.8%	0.8%
Between-Run	0.0%	0.1%	0.0%	0.0%	0.0%	0.0%	0.0%	0.0%	0.0%
Between-Day	0.9%	0.3%	0.5%	0.4%	0.2%	0.4%	0.6%	0.2%	0.3%
Between-Lot	2.0%	1.1%	2.2%	1.6%	1.3%	0.7%	2.3%	1.0%	0.7%
Total Precision	2.5%	1.4%	2.5%	1.9%	1.6%	1.1%	2.7%	1.3%	1.1%

510(k) Summary

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Instrument ID: SM94
Variation Source	Control 1	Control 2	Patient 1	Patient 2	Patient 3	Patient 4	QC 1	QC 2	QC 3
ConcentrationHbA1c (NGSP%)	36.4	79.2	32.9	49.0	65.1	108.0	34.5	85.4	137.9
Repeatability	1.7%	1.2%	1.4%	1.4%	1.3%	1.1%	1.7%	1.3%	1.1%
Between-Run	0.0%	0.4%	0.0%	0.0%	0.3%	0.0%	0.0%	0.1%	0.1%
Between-Day	1.0%	0.3%	0.8%	0.7%	0.4%	0.4%	0.3%	0.5%	0.4%
Between-Lot	2.1%	0.3%	2.6%	1.0%	0.0%	0.4%	2.5%	0.1%	0.6%
Total Precision	2.9%	1.4%	3.1%	1.8%	1.4%	1.2%	3.1%	1.4%	1.3%

Table 8: Instrument 2 (% CV by Sample (IFCC Units- mmol/mol))

Table 9: Instrument 3 (% CV by Sample (IFCC Units- mmol/mol)

Variation Source	Instrument ID: SM95
	Control 1	Control 2	Patient 1	Patient 2	Patient 3	Patient 4	QC 1	QC 2	QC 3
ConcentrationHbA1c (NGSP%)	36.0	79.1	32.7	48.9	64.9	107.7	34.1	84.8	137.6
Repeatability	1.6%	1.2%	1.8%	1.7%	1.3%	1.1%	1.6%	1.2%	1.0%
Between-Run	0.0%	0.0%	0.0%	0.0%	0.0%	0.0%	0.0%	0.0%	0.0%
Between-Day	1.0%	0.6%	0.8%	0.8%	0.7%	0.5%	0.7%	0.5%	0.5%
Between-Lot	2.6%	1.2%	2.6%	2.3%	1.6%	1.3%	2.9%	1.2%	1.2%
Total Precision	3.2%	1.8%	3.3%	2.9%	2.1%	1.8%	3.4%	1.7%	1.7%

Table 10: Instruments Combined (% CV by Sample (IFCC Units- mmol/mol)

Variation Source	All Instruments
	Control 1	Control 2	Patient 1	Patient 2	Patient 3	Patient 4	QC 1	QC 2	QC 3
ConcentrationHbA1c (NGSP %)	36.3	79.2	33.0	49.1	65.1	107.9	34.3	85.1	137.8
Repeatability	1.5%	1.1%	1.5%	1.4%	1.2%	1.0%	1.5%	1.1%	1.0%
Between-Run	0.0%	0.0%	0.0%	0.0%	0.1%	0.0%	0.0%	0.0%	0.0%
Between-Day	1.0%	0.4%	0.7%	0.6%	0.5%	0.4%	0.6%	0.4%	0.4%
Between-Instrument	0.0%	0.0%	0.0%	0.0%	0.0%	0.0%	0.0%	0.0%	0.0%
Between-Lot	2.2%	1.0%	2.5%	1.7%	1.2%	0.9%	2.6%	0.9%	0.9%
Total Precision	2.9%	1.5%	3.0%	2.3%	1.7%	1.4%	3.1%	1.5%	1.4%

b. Linearity

A linearity study was performed per CLSI EP06-A: Evaluation of the Linearity of Quantitative Measuring Procedures; A Statistical Approach. Linearity across the reportable range was performed using low (3.5%HbA1c) and high (20%HbA1c) EDTA whole blood patient samples. These samples 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.

% HbA1c (NGSP) using the D-100™ HbA1c test has been demonstrated linear from 3.5 - 20.0% HbA1c with the maximum measured difference of

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± 0.09% between the predicted 1* and 2 ° order results as shown in Table 11 below.
Mmol/mol HbA1c (IFCC) has been demonstrated as linear from 15 – 195 mmolmol with the maximum measured difference of ± 0.9% (or +/- 0.94mmol/mol) as shown in Table 12 below.

Sample Pool	Predicted 1stOrder	Predicted 2ndOrder	Difference
Control, Level1	3.50	3.43	0.07
Level 2	5.16	5.14	0.02
Level 3	6.82	6.85	-0.03
Level 4	8.48	8.54	-0.06
Level 5	10.13	10.21	-0.08
Level 6	11.79	11.88	-0.09
Level 7	13.44	13.53	-0.09
Level 8	15.09	15.17	-0.08
Level 9	16.74	16.79	-0.05
Level 10	18.39	18.41	-0.02
High, Level 11	20.04	20.01	0.03

Table 11: Results of Linearity Study (NGSP %)

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Table 12: Results of Linearity Study (IFCC mmol/mol)

Sample Pool	Predicted 1stOrder	Predicted 2ndOrder	Difference
Control, Level 1	14.78	13.85	0.92
Level 2	32.93	32.56	0.37
Level 3	51.07	51.12	-0.28
Level 4	69.19	69.55	-0.36
Level 5	87.30	87.83	-0.54
Level 6	105.38	105.98	-0.60
Level 7	123.46	123.99	-0.53
Level 8	141.51	141.86	-0.34
Level 9	159.55	159.59	-0.04
Level 10	177.57	177.18	0.39
High, Level 11	195.58	194.63	0.94

Method Comparison ﻥ

A Method comparison study was performed per CLSI EP09-A2 IR, Method Comparison and Bias Estimation Using Patient Samples. 129 variant-free whole blood EDTA samples ranging from 3.5% to 20.0% HbA1c were evaluated using the D-100™ HbA1c on the D-100TM Hemoglobin Testing System. Samples were tested in a single determination over a 4 day period. The results were compared to testing performed at a secondary NGSP SRL reference laboratory using a cleared HPLC-based HbA1c assay. The distribution of samples spanned the measuring interval listed in Table 13.

Hemoglobin A1c level	n	% Samples tested
≤ 5%	6	4.7
5 - 6%	17	13.2
6 - 6.5%	31	24.0
6.5 - 7%	33	25.6
7 - 8%	20	15.5
8 - 9%	11	8.5
> 9%	11	8.5
Total samples	129	100

Table 13: Distribution of samples

Deming (weighted) and Passing-Bablok regression analyses were performed for the D-100™ HbA1c versus the NGSP SRL reference method. Deming (weighted), Passing

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Bablok and Linear regression analyses were performed for the D-100™ HbA1c on the D-100 Hemoglobin Testing System versus the reference G8 HPLC method are summarized in Table 14.

Table 14: Summary of Method Comparison Results

	y-Intercept	95% CI	Slope	95% CI
Deming	0.0223	-0.0684 – 0.1131	0.9867	0.9736 – 0.9999
Passing-Bablok	-0.0091	-0.0803 – 0.0763	0.9909	0.9789 – 1.0026

Image /page/12/Figure/3 description: This image is a scatter plot with a Deming fit. The x-axis is labeled "% HbA1c, NGSP SRL" and the y-axis is labeled "% HbA1c, D100 HbA1C". The data points are clustered tightly around the Deming fit line, and the R-squared value is 0.9996.

Figure 1: Scatter Plot using Deming Fit, %HbA1c, NGSP SRL vs. D-100 HbA1c.

(1) The following biases between D-100 HbA1c versus NGSP SRL Method (Reference method) were observed in Table 15.

% HbA1c - DecisionLevel	Bias	% Bias
5.0	-0.047	-0.85

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6.5	-0.066	-0.98
8.0	-0.090	-1.11
12.0	-0.190	-1.57

Total Error Decision Levels

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: %TE=|%Bias| + 1.96 CV (1 + %Bias). The results are presented in Table 16.

Table 16: Total Error Estimation

% A1c – Decision Level	% Bias	% CV	% TE
5.0	-0.85	1.7	4.2
6.5	-0.98	1.5	3.9
8.0	-1.11	1.3	3.6
12.0	-1.57	1.2	3.9

d. Traceability, Stability, Expected Values (calibrators)
The D-100 HbA1c test standardization is traceable to the International Federation of Clinical Chemistry (IFCC) reference calibrators. The D-100 HbA1c assay is NGSP certified. The NGSP certification expires in one year. See NGSP website for current certification at http://www.ngsp.org. The derived results of (%) from the NGSP correlation are calculated from the individual quantitative results for Hemogloblin A1c (HbA1c). The International Federation of Clinical Chemistry (IFCC) units of mmol/mol are calculated using the Master Equation NGSP (%) = 0.09148 x 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).

Calibrator Materials:

Value assignment for D-100™ HbA1c Calibrators are traceable to IFCC reference method and can be transferred to DCCT/NGSP by calculation.

Stability:

Shelf life claims: Un-opened calibrators can be stored at 2-8°C until the expiration date or for 24 months.

Open-vial claims: The recommended storage condition for in-use calibrators is one day. On-board stability for the D-100 HbA1c calibrator pack and reagents demonstrated 90 days stability on the D-100 Hemoglobin Testing System.

e. Analytical specificity:

Endogenous Interference i.)
An Endogenous Interference study was performed per CLSI EP07-A2, Interference Testing in Clinical Chemistry. Two EDTA whole blood sample pools were evaluated using a low level whole blood sample with a concentration ~6.5%HbA1c and a high level whole blood sample with a concentration of HbA1c of ~8.0%.

Conjugated bilirubin, unconjugated bilirubin and glucose, available in pure

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form, were obtained and stock solutions prepared at 10x the intended test concentration. The 10x stock solution of the test substance was pipetted into a low whole blood sample pool (at ~6.5% HbA1c) and a high whole blood sample pool (~8.0% HbA1c), making the test pool. Ten replicates of each pool prepared with the test and control samples were analyzed using the D-100™ HbA1c on the D-100™Hemoglobin Testing System.

Rheumatoid factor, lipemia and total protein were not available as pure standards therefore serum samples with known concentration of these compounds were used. The test pool was prepared by mixing the serum sample known to have a high test substance concentration with a whole blood non-variant sample such that the concentration of test substance in the final mixture would be at the desired level. Ten replicates of each pool prepared with the test and control samples were analyzed using the D-100™ HbA1c on the D-100™ Hemoglobin Testing System.

Significant interference was defined as a ± 7% change in %HbA1c value from the control. Results in Table 17 showed no significant interference up to the stated concentrations.

	Concentration
Endogenous substance	Conventional (US)units	SI Units
Lipemia (Intralipid)	6000 mg/dL	60 g/L
Conjugated bilirubin	60 mg/dL	712 $\mu$ mol/L
Unconjugated bilirubin	60 mg/dL	1026 $\mu$ mol/L
Glucose	2000 mg/dL	111 mmol/L
Rheumatoid factor	750 IU/mL	750 kIU/mL
Total protein	21 g/dL	210 g/L

Table 17: Endogenous Interference Study Results

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 low level whole blood sample with a concentration ~6.5%HbA1c and a high level whole blood sample with a concentration of ~8.0%HbA1c. Test samples were prepared by spiking each drug at the interferent concentration shown in Table 18. Ten replicates of each drug prepared with the test and control samples were analyzed using the D-100™ HbA1c on the D-100™ 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 Table 18 on the following page.

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Potential DrugInterferent	Highest Level Tested showing no SignificantInterference
	Conventional (US)units	SI units
Acetylcysteine	166 mg/dL	10.2 mmol/L
Ampicillin-Na	1000 mg/dL	28.65 mmol/L
Ascorbic acid	300 mg/dL	17.05 mmol/L
Cefoxitin	2500 mg/dL	58.55 mmol/L
Heparin	5000 U/L	5000 U/L
Levodopa	20 mg/dL	1015 µmol/L
Methyldopa	20 mg/dL	948 µmol/L
Metronidazole	200 mg/dL	11.7 mmol/L
Doxycyclin	50 mg/dL	1124 µmol/L
Acetylsalicylic acid	1000 mg/dL	55.51 mmol/L
Rifampicin	64 mg/L	78 µmol/L
Cyclosporine	5 mg/L	4 µmol/L
Acetaminophen	200 mg/L	1323 µmol/L
Ibuprofen	500 mg/L	2427 µmol/L
Theophylline	100 mg/L	556 µmol/L
Phenylbutazone	400 mg/L	1299 µmol/L

Table 18: Drug Interference Study Results

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 hemogloblin (Hb), Carbamylated hemoglobin (Hb) and Labile HbA1c were evaluated using a low level whole blood EDTA sample with a concentration ~6.5%HbA1c and a high level whole blood EDTA sample with a concentration of

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~8.0% HbA1c. The potentially interfering hemoglobin derivatives were spiked into the low and high level blood samples and each sample was analyzed using ten replicates each in the same analytical run on the D-100™ Hemoglobin Testing System with the D-100™HbA1c.

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 5% does not interfere with this assay.
Labile A1c- up to 1200 mg/dL of glucose does not interfere with this . assay.

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

iv.) Hemoqlobin Variant Study:

A Hemoglobin Variant study was performed using specific variant samples known to contain hemoglobin variants S. C. E. D. A2 and F. Two whole blood EDTA patient samples containing an HbA1c ~6.5% and ~ 8% and the appropriate hemoglobin variant were tested. Testing of the samples containing hemoglobin variants S. C. E. D. A2 and F were performed in duplicate. Testing of the samples was performed using the D-100™ HbA1c on the D-100™ Hemoglobin Testing System and compared to results obtained by a NGSP reference method that has been demonstrated to be free from the hemoglobin interferent. Table 19 contains the number of samples, range of samples and concentration of samples used in the Hemoglobin Variant Study. Table 20 contains the results for the Hemoglobin Variant study bias.

HemoglobinVariant	n	Range in % AbnormalVariant	Range in %HbA1cConcentration
HbS	20	28.7 - 40.2	5.6 - 9.6
HbC	20	34.4 - 44.1	5.0 - 10.7
HbD	20	36.6 - 43.4	5.8 - 8.6
HbE	20	25.5 - 32.5	5.9 - 8.3
HbA2	25	5.0 - 13.3	5.0 - 14.5
HbF	30	4.1 - 30.2	4.4 - 14.4

Table 19: Variant samples used in Hemoglobin Variant Study

Table 20: Hemoglobin Variant Study Bias Results

HemoglobinRelative % Bias to Comparative Method
Variant	Relative %Bias (Range of %Bias) forHbA1c~6.5%	Relative %Bias (Range of %Bias)for HbA1c~9.0%
HbS	-0.6 (-5.8 to 5.5)	-1.5 (-3.3 to -0.1)
HbC	-1.3 (-4.0 to 1.3)	-3.9 (-5.5 to -2.4)
HbD	-4.7 (-6.7 to -1.1)	-4.4 (-6.3 to -2.4)
HbE	-2.7 (-6.7 to 1.6)	-1.3 (-2.0 to -0.6)
HbA2	-1.3 (-5.1 to 0.5)	3.4 (2.8 to 4.1)
HbF	-2.3 (-4.1 to -0.7)	-3.5 (-4.2 to -2.8)

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2. Matrix comparison

The data supports the use of the following blood collection tubes with the D-100™ HbA1c test in Table 21.

Table 21: Anticoagulant
K2-EDTA
K3-EDTA
Potassium Oxalate/Sodium Fluoride
Sodium Citrate
Sodium Heparin
Lithium Heparin

3. Expected Values/Reference Range

Hemoglobin A1c expected values range was cited from American Diabetes Association Standards of Medical Care in Diabetes 2010, 33 (Supplement 1), S62-S69 for Diagnosis of Diabetes are presented in Table 22.

Table 22: Hemoglobin A1c Expected Values

Hemoglobin A1c	Suggested Diagnosis
NGSP %	IFCC mmol/mol
>6.5	>47	Diabetic
5.7 - 6.4	39-46	Pre-Diabetic
<5.7	<39	Non-Diabetic

Conclusion:

The information and data in this 510(k) document demonstrate that the D-100™ HbA1c test as performed on the D-100™Hemoglobin Testing System is an accurate, reliable, precise test that correlates well with current cleared methods and NGSP standardized testing for the quantitation of HbA1c. The contents of this submission demonstrates that the D-100™ HbA1c test as performed on the D-100™ Hemoglobin Testing System is substantially equivalent to its predicate device, VARIANT II TURBO HbA1c Kit - 2.0 and, therefore, safe and effective for its intended use. The performance criteria as stipulated by the Special Controls requirements for HbA1c systems that diagnose diabetes have clearly been met. The D-100™ HbA1c must be found to be substantially equivalent to the predicate and, therefore, cleared by the agency for the intended use requested.

Regulation Number and Section

§ 862.1373 Hemoglobin A1c test system.

(a)
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.(b)
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.