Afinion HbA1c Dx
Device Facts
| Record ID | K180296 |
|---|---|
| Device Name | Afinion HbA1c Dx |
| Applicant | Alere Technologies AS |
| Product Code | PDJ · Clinical Chemistry |
| Decision Date | May 7, 2018 |
| Decision | SESE |
| Submission Type | Traditional |
| Regulation | 21 CFR 862.1373 |
| Device Class | Class 2 |
Intended Use
The Afinion HbA1c Dx is an in vitro diagnostic test for quantitative determination of glycated hemoglobin (% hemoglobin A1c, HbA1c) in human venous and capillary whole blood. This test is to be used as an aid in the diagnosis of diabetes and as an aid in identifying patients who may be at risk for developing diabetes. The measurement of % HbA1c is recommended as a marker of long-term metabolic control in persons with diabetes mellitus.
Device Story
Afinion™ HbA1c Dx is a fully automated boronate affinity assay for quantitative determination of % HbA1c in human whole blood. Used with the Alere Afinion™ AS100 Analyzer, the device processes samples collected via an integrated sampling device. The analyzer automatically dilutes the sample, releases hemoglobin from erythrocytes, and precipitates it. The mixture is transferred to a blue boronic acid conjugate that binds to glycated hemoglobin cis-diols. The mixture is filtered; the analyzer measures reflectance of the precipitate on the membrane as blue (glycated) and red (total) intensities. A calculated ratio, proportional to % HbA1c, is displayed. Used in clinical settings by healthcare professionals, the output provides a marker for long-term metabolic control, aiding in diabetes diagnosis and risk identification. The device is factory-calibrated, requiring no user calibration.
Clinical Evidence
Performance evaluated via bench testing and method comparison. Precision studies (internal and external) assessed repeatability and reproducibility across multiple sites, analyzers, and lots, with total CVs generally <2%. Method comparison study (n=120) against an NGSP secondary reference laboratory (Tosoh G8 HPLC) showed strong correlation (r=0.990-0.991). Interference studies evaluated endogenous substances, drugs, and hemoglobin variants; significant negative interference identified with fetal hemoglobin (HbF).
Technological Characteristics
Boronate affinity assay; reflectance-based measurement of blue boronic acid conjugate and red total hemoglobin. Polyethersulfone membrane filter. Factory-calibrated. Dimensions/form factor: cartridge-based system for Afinion AS100 analyzer. Connectivity: standalone analyzer. Software: embedded firmware. Sterilization: not specified.
Indications for Use
Indicated for use by healthcare professionals in clinical laboratories to quantitatively measure % HbA1c in human venous and capillary whole blood to aid in the diagnosis of diabetes, identify patients at risk for developing diabetes, and monitor long-term metabolic control in patients with diabetes mellitus. Contraindicated for patients with abnormal amounts of fetal hemoglobin (HbF), including Hereditary Persistence of Fetal Hemoglobin, due to significant negative interference.
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 Diagnostics Cobas C501 Tina-Quant HbA1c cDx Gen 3 assay (k121610)
Related Devices
- K121366 — HEMOCUE HBA1C 501 · Infopia Co, Ltd. · Sep 7, 2012
- K214117 — Afinion HbA1c, Afinion 2 and Alere Afinion AS100 Analyzer · Abbott Diagnostics Technologies AS · Sep 27, 2023
Submission Summary (Full Text)
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# 510(k) SUBSTANTIAL EQUIVALENCE DETERMINATION DECISION SUMMARY ASSAY ONLY TEMPLATE
A. 510(k) Number:
k180296
B. Purpose for Submission:
Addition of a diagnostic claim to an existing device
C. Measurand:
Glycosylated Hemoglobin (HbA1c)
D. Type of Test:
Quantitative boronate affinity assay
E. Applicant:
Alere Technologies AS
F. Proprietary and Established Names:
Afinion HbA1c Dx
G. Regulatory Information:
| Description | Classification | Regulation | Product Code | Panel |
| --- | --- | --- | --- | --- |
| Hemoglobin A1c Test System | Class II | 21 CFR 862.1373 | PDJ | Chemistry (75) |
H. Intended Use:
1. Intended use(s):
See indications for use below.
2. Indication(s) for use:
The Afinion HbA1c Dx is an in vitro diagnostic test for quantitative determination of glycated hemoglobin (% hemoglobin A1c, HbA1c) in human venous and capillary whole blood. This test is to be used as an aid in the diagnosis of diabetes and as an aid in
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identifying patients who may be at risk for developing diabetes. The measurement of % HbA1c is recommended as a marker of long-term metabolic control in persons with diabetes mellitus.
3. Special conditions for use statement(s):
For prescription use only.
For in vitro diagnostic use.
This device has significant negative interference with fetal hemoglobin (HbF). HbA1c results are invalid for patients with abnormal amounts of HbF including those with known Heredity Persistence of Fetal Hemoglobin.
This test should not be used to diagnose:
- Diabetes during pregnancy
- Patients with an elevated fetal hemoglobin (HbF > 10%) such as hereditary persistence of fetal hemoglobin (HPFH)
- Patients with a hemoglobinopathy but normal red cell turnover (e.g. sickle cell trait)
- Patients with abnormal red cell turnover (e.g. anemias from hemolysis and iron deficiency)
- Patients with iron deficiency and hemolytic anemia, various hemoglobinopathies, thalassemias, hereditary spherocytosis, malignancies, and severe chronic hepatic and renal disease
- Patients that have received a blood transfusion within the past 3 weeks
- Patients that have received cancer chemotherapy within the past 3 weeks
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.
HbA1c testing should not replace glucose testing for type 1 diabetes, in pediatric patients and in pregnant women.
Coagulated or hemolyzed samples cannot be used with Afinion HbA1c Dx. Samples with > 14% hemolysis (2000 mg/dL) may return an information code.
If the sample has a hemoglobin value below 6 g/dL or above 20 g/dL, no test result will be reported and an information code will be displayed.
4. Special instrument requirements:
Afinion AS100 analyzer
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I. Device Description:
The Afinion HbA1c Dx is a fully automated boronate affinity assay for the determination of the percentage of hemoglobin A1c in human venous and capillary whole blood. Each assay cartridge contains blue boronic acid conjugate, a tube with a polyethersulfone membrane, and washing solution (morpholine buffered sodium chloride solution with detergents and preservative). Each cartridge is labeled with a unique barcode.
The Afinion HbA1c Dx is factory calibrated and intended for use with the previously cleared Afinion AS100 Analyzer (k050574, k110056) and Afinion HbA1c Controls (k050574).
J. Substantial Equivalence Information:
1. Predicate device name(s):
Roche Diagnostics Cobas C501 Tina-Quant HbA1c cDx Gen 3 assay
2. Predicate 510(k) number(s):
k121610
3. Comparison with predicate:
| Similarities | | |
| --- | --- | --- |
| Item | Candidate Device Afinion HbA1c Dx | Predicate Cobas c 501 Tina-Quant HbA1c Dx Gen. 3 assay (k121610) |
| Intended Use | Quantitative determination of hemoglobin A1c for the diagnosis of diabetes and as an aid in identifying patients who may be at risk for developing diabetes | Same |
| Units of measurement | % HbA1c (DCCT/NGSP) | Same |
| Storage requirements | 2-8°C until expiration date | Same |
| Controls | Provided by manufacturer | Same |
| Traceability | Traceable to the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) and Diabetes Control and Complications Trial (DCCT) reference method. Certified via the National Glycohemoglobin | Same |
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| Similarities | | |
| --- | --- | --- |
| Item | Candidate Device Afinion HbA1c Dx | Predicate Cobas c 501 Tina-Quant HbA1c Dx Gen. 3 assay (k121610) |
| | Standardization Program (NGSP) | |
| Differences | | |
| --- | --- | --- |
| Item | Candidate Device Afinion HbA1c Dx | Predicate Cobas c 501 Tina-Quant HbA1c Dx Gen. 3 assay (k121610) |
| Intended use sites | For use by health care professionals in clinical laboratories and point of care settings | For use by health care professionals in clinical laboratories |
| Assay principle | Boronate affinity assay. Hemoglobin determination by reflectance measurements of blue conjugate of glycated hemoglobin and red total hemoglobin. | HbA1c determination by turbidimetric inhibition immunoassay method. Hemoglobin determination by converting to a derivative with a characteristic absorption spectrum which is measured bichromatically. |
| Sample types | Anticoagulated venous whole blood (K2-EDTA), and capillary whole blood (fingerstick) | Anticoagulated venous whole blood (K2-EDTA, K3-EDTA, Li-Heparin, Na-Heparin, NaF/Na2-EDTA, NaF/K-oxalate, KF/ Na2-EDTA) |
| Analyzer | Alere Afinion AS100 | Cobas c 501 |
| Calibrators | Factory calibrated, no user calibration required | User calibration using calibrators provided by manufacturer |
| Assay range | 4.00 – 15.00 % HbA1c | 4.2 – 20.1 % HbA1c |
# K. Standard/Guidance Document Referenced (if applicable):
Clinical and Laboratory Standards Institute (CLSI) EP5-A3 Evaluation of Precision Performance of Quantitative Measurement Methods, Approved Guideline—Third Edition.
CLSI EP7-A2 Interference Testing in Clinical Chemistry, Approved Guideline -- Second Edition
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CLSI EP5-A3 Evaluation of Precision of Quantitative Measurement Procedures- Approved Guideline -Third Edition
ISO 14971 Second edition 2007-03-01, Medical devices - application of risk management to medical devices
EN ISO 14971:2012 Medical devices, Application of risk management to medical devices
## L. Test Principle:
A blood sample is collected with the integrated sampling device, which is then inserted into the Alere HbA1c Dx test cartridge. The cartridge/sampling device combination is then placed into the cartridge chamber of the Alere Afinion AS100 Analyzer. The sample is automatically diluted and mixed with a solution that releases hemoglobin from the erythrocytes. After the hemoglobin is precipitated, the sample mixture is transferred to a blue boronic acid conjugate which binds to the cis-diols of glycated hemoglobin. This reaction mixture is soaked through a filter membrane and all precipitated hemoglobin, conjugate-bound and unbound (i.e. glycated and nonglycated hemoglobin) remains on the membrane. Excess conjugate is removed with a washing reagent. The analyzer measures the reflectance of the precipitate on the membrane as blue (glycated hemoglobin) and red (total hemoglobin) color intensities. The analyzer calculates a ratio proportional to the percentage of HbA1c in the sample and displays as the $\%$ HbA1c (DCCT/NGSP).
## M. Performance Characteristics (if/when applicable):
### 1. Analytical performance:
#### a. Precision/Reproducibility:
Internal precision study using venous whole blood
An internal precision study was performed according to CLSI guideline EP05-A3. Four $\mathrm{K}_2$ -EDTA venous whole blood samples at concentrations near $5\%$ , $6.5\%$ , $8\%$ and $12\%$ HbA1c were analyzed by eleven operators in duplicate, twice a day, with three lots of reagents, over 20 days each, on nine Afinion AS100 analyzers (three lots per analyzer, three analyzers per sample). For each sample, there were 720 measurements. Results are shown in the tables below:
Analyzer 1 $(\mathrm{n} = 240)$
| Level | Mean %HbA1c | Repeatability | | Between run | | Between day | | Between lot | | Total | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| | | SD | %CV | SD | %CV | SD | %CV | SD | %CV | SD | %CV |
| Low | 5.13 | 0.067 | 1.31 | 0.017 | 0.33 | 0.029 | 0.56 | 0.037 | 0.72 | 0.084 | 1.63 |
| Threshold | 6.53 | 0.086 | 1.31 | 0.000 | 0.00 | 0.039 | 0.60 | 0.064 | 0.98 | 0.114 | 1.74 |
| Medium | 8.09 | 0.082 | 1.01 | 0.000 | 0.00 | 0.045 | 0.56 | 0.019 | 0.23 | 0.095 | 1.17 |
| High | 11.33 | 0.100 | 0.88 | 0.000 | 0.00 | 0.035 | 0.31 | 0.105 | 0.93 | 0.149 | 1.32 |
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Analyzer 2 $(n = 240)$ :
| Level | Mean | Repeatability | | Between run | | Between day | | Between lot | | Total | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| | %HbA1c | SD | %CV | SD | %CV | SD | %CV | SD | %CV | SD | %CV |
| Low | 5.11 | 0.061 | 1.19 | 0.000 | 0.00 | 0.034 | 0.67 | 0.046 | 0.89 | 0.084 | 1.63 |
| Threshold | 6.53 | 0.070 | 1.07 | 0.019 | 0.30 | 0.044 | 0.67 | 0.064 | 0.98 | 0.106 | 1.62 |
| Medium | 8.01 | 0.060 | 0.75 | 0.010 | 0.13 | 0.038 | 0.48 | 0.042 | 0.52 | 0.083 | 1.04 |
| High | 11.17 | 0.086 | 0.77 | 0.015 | 0.13 | 0.060 | 0.54 | 0.094 | 0.84 | 0.142 | 1.27 |
Analyzer 3 $(n = 240)$ :
| Level | Mean | Repeatability | | Between run | | Between day | | Between lot | | Total | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| | %HbA1c | SD | %CV | SD | %CV | SD | %CV | SD | %CV | SD | %CV |
| Low | 5.18 | 0.053 | 1.02 | 0.007 | 0.14 | 0.030 | 0.58 | 0.041 | 0.79 | 0.074 | 1.43 |
| Threshold | 6.58 | 0.066 | 1.00 | 0.000 | 0.00 | 0.038 | 0.58 | 0.035 | 0.53 | 0.084 | 1.27 |
| Medium | 8.08 | 0.086 | 1.06 | 0.035 | 0.43 | 0.052 | 0.64 | 0.047 | 0.58 | 0.116 | 1.43 |
| High | 11.30 | 0.114 | 1.00 | 0.023 | 0.21 | 0.031 | 0.28 | 0.098 | 0.87 | 0.155 | 1.37 |
All analyzers combined $(n = 720)$ :
| Level | Mean | Repeatability | | Between run | | Between day | | Between lot | | Between analyzer | | Total | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| | %HbA1 | SD | %CV | SD | %CV | SD | %CV | SD | %CV | SD | %CV | SD | %CV |
| Low | 5.14 | 0.061 | 1.18 | 0.000 | 0.00 | 0.031 | 0.60 | 0.041 | 0.81 | 0.030 | 0.58 | 0.08 | 1.64 |
| Threshold | 6.55 | 0.074 | 1.13 | 0.000 | 0.00 | 0.040 | 0.62 | 0.056 | 0.85 | 0.000 | 0.00 | 0.09 | 1.51 |
| Medium | 8.06 | 0.077 | 0.95 | 0.016 | 0.20 | 0.045 | 0.56 | 0.038 | 0.47 | 0.039 | 0.49 | 0.10 | 1.31 |
| High | 11.26 | 0.101 | 0.89 | 0.014 | 0.12 | 0.044 | 0.39 | 0.099 | 0.88 | 0.059 | 0.52 | 0.16 | 1.42 |
# External precision study using venous whole blood
An external precision study was performed at three point-of-care moderate complexity laboratory sites in the US. Four $\mathrm{K}_2$ -EDTA venous whole blood samples at concentrations near $5\%$ , $6.5\%$ , $8\%$ and $12\%$ HbA1c were analyzed by two to three operators per site using six Afinion AS100 analyzers and three lots of reagent. Four replicates were analyzed twice a day for 10 days for a total of 240 measurements per sample at each site. The data was analyzed according to CLSI EP05-A3 and results are shown in the table below:
All sites combined:
| Level | Mean | Repeatability | | Between run | | Between day | | Between lot | | Between analyzer | | Total | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| | %HbA1 | SD | %CV | SD | %CV | SD | %CV | SD | %CV | SD | %CV | SD | %CV |
| Low | 5.02 | 0.061 | 1.21 | 0.029 | 0.57 | 0.02 | 0.44 | 0.046 | 0.92 | 0.030 | 0.58 | 0.085 | 1.78 |
| Threshold | 6.41 | 0.071 | 1.11 | 0.030 | 0.48 | 0.02 | 0.40 | 0.047 | 0.74 | 0.000 | 0.00 | 0.095 | 1.48 |
| Medium | 8.18 | 0.090 | 1.10 | 0.019 | 0.23 | 0.02 | 0.28 | 0.042 | 0.51 | 0.039 | 0.49 | 0.104 | 1.36 |
| High | 12.09 | 0.118 | 0.97 | 0.046 | 0.38 | 0.03 | 0.25 | 0.033 | 0.27 | 0.059 | 0.52 | 0.134 | 1.22 |
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# Capillary blood (fingerstick) precision:
Capillary blood (fingerstick) precision estimates are based on a combination of three separate studies as described below:
i. Capillary blood (fingerstick) precision estimates for repeatability and between-lot were calculated from measurements obtained during the method comparison study. Ten intended use operators analyzed duplicate fingerstick samples from 170 subjects with three reagent lots in total (two lots per site) on five Afinion AS100 analyzers at three sites. The samples were grouped into four $\% \mathrm{HbA1c}$ concentration intervals (4.00-5.99, 6.00-6.99, 7.00-9.99, 10.00-15.00) and analyzed according to CLSI guideline EP05-A3. Results are presented in the table below:
| Level | N | Minimum %HbA1c | Maximum %HbA1c | Average %HbA1c | Repeatability | | Between lot | | Total | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| | | | | | SD | %CV | SD | %CV | SD | %CV |
| Low | 45 | 4.77 | 5.99 | 5.42 | 0.082 | 1.52 | 0.065 | 1.20 | 0.105 | 1.93 |
| Threshold | 68 | 6.00 | 6.98 | 6.46 | 0.088 | 1.36 | 0.027 | 0.42 | 0.092 | 1.42 |
| Medium | 51 | 7.02 | 9.94 | 7.93 | 0.107 | 1.35 | 0.000 | 0.00 | 0.107 | 1.35 |
| High | 6 | 10.07 | 11.52 | 10.72 | 0.067 | 0.62 | 0.008 | 0.07 | 0.067 | 0.62 |
ii. To capture repeatability, between-instrument, and between-operator precision estimates, an external study was performed at three point-of-care moderate complexity laboratory sites in the US. Samples in four $\% \mathrm{HbA1c}$ concentration intervals (4.00-5.99, 6.00-6.99, 7.00-9.99, 10.00-15.00) from five or six subjects per interval were analyzed by three operators per site using two Afinion AS100 analyzers. Six fingerstick samples (two fingerstick samples from each subject were collected and measured per operator) were analyzed for a total of 30 or 36 measurements (M) per sample level at each site ( $n = 90$ or $n = 96$ measurements across sites). The results from all sites combined are shown in the table below.
| Level | N (M) | Average %HbA1c | Repeatability | | Between Instrument | | Between Operator | | Total | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| | | | SD | %CV | SD | %CV | SD | %CV | SD | %CV |
| Low | 15 (90) | 5.33 | 0.074 | 1.39 | 0.008 | 0.14 | 0.00 | 0.00 | 0.075 | 1.40 |
| Threshold | 15 (90) | 6.51 | 0.081 | 1.25 | 0.019 | 0.30 | 0.00 | 0.00 | 0.083 | 1.28 |
| Medium | 16 (96) | 8.32 | 0.092 | 1.10 | 0.000 | 0.00 | 0.017 | 0.21 | 0.093 | 1.12 |
| High | 15 (90) | 12.20 | 0.139 | 1.14 | 0.057 | 0.47 | 0.00 | 0.00 | 0.150 | 1.23 |
iii. Between-Run and Between-Day precision estimates for capillary blood were transferred from the internal venous whole blood precision study described above.
The combined precision estimates (%CV) from the three studies are shown in the table below.
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| Level | Fingerstick Accuracy Precision | | Internal Venous Precision Study | | Fingerstick Precision Study | | | Total %CV |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| | Repeatability | Between Lot | Between Run | Between Day | Repeatability | Between Instrument | Between Operator | |
| Low | 1.52 | 1.2 | 0.00 | 0.60 | - | 0.14 | 0.00 | 2.03 |
| Threshold | 1.36 | 0.42 | 0.00 | 0.62 | - | 0.30 | 0.00 | 1.58 |
| Medium | 1.35 | 0 | 0.20 | 0.56 | - | 0.00 | 0.21 | 1.49 |
| *High | - | 0.07 | 0.12 | 0.39 | 1.14 | 0.47 | 0.00 | 1.30 |
*This estimate of the Total %CV for High HbA1c (≥ 10%) is based on the within run component of variance estimated in the Fingerstick Precision Study since the precision study has a larger sample size than the Fingerstick Accuracy Precision Study.
b. Linearity/assay reportable range:
Linearity was previously established for the range of 4.00 – 15.00 % HbA1c (DCCT/NGSP) in k050574.
c. Traceability, Stability, Expected values (controls, calibrators, or methods):
The Afinion HbA1c Dx assay is 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. Traceability is verified annually through the IFCC Whole Blood Monitoring Program.
Test results are calculated in mmol/mol (IFCC). HbA1c values are further reported according to the National Glycohemoglobin Standardization Program (NGSP) recommendations at DCCT (Diabetes Control and Complications Trial) level. The IFCC Master Equation for the relationship between the IFCC-Reference Method (RM) and the NGSP-Designated Comparison Method (DCM) is used for conversion of the test results:
$$
\mathrm {NGSP - HbA1c} (\%) = \mathrm {DCCT - HbA1c} (\%) = 0.09148 \times \mathrm {IFCC - HbA1c} (\mathrm {mmol/mol}) + 2.152
$$
d. Detection limit:
The Limit of Quantification was previously established as 4.0% HbA1c in k050574.
e. Analytical specificity:
i. Exogenous and endogenous interference
Interference from 20 drug substances and six endogenous compounds was evaluated using K₂-EDTA venous whole blood samples with low (~6.5%) and high (≥ 8.5%) HbA1c levels. Test samples were prepared by spiking each drug or endogenous substance into the low and high samples and comparing to control
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samples containing no drug. The test and control samples were tested in 10 replicates using two lots of reagent. The sponsor defined significant interference as greater than or equal to $7\%$ difference from the expected concentration. Results demonstrated that no significant interference was observed with the following substances up to the listed concentrations:
| Substance | Highest concentration tested with no significant interference |
| --- | --- |
| Acetaminophen | 20 mg/dL |
| Acetylcysteine | 166 mg/dL |
| Acetylsalicylic acid | 100 mg/dL |
| Ampicillin | 100 mg/dL |
| Ascorbic acid | 30 mg/dL |
| Bilirubin -conjugated | 60 mg/dL |
| Bilirubin - unconjugated | 60 mg/dL |
| Cefoxitin | 250 mg/dL |
| Cyclosporine A | 0.5 mg/dL |
| Cyclosporine C | 0.5 mg/dL |
| Doxycyclin | 5 mg/dL |
| Glucose | 1000 mg/dL |
| Glyburide | 0.20 mg/dL |
| Heparin | 5000 U/L |
| Ibuprofen | 50 mg/dL |
| Intralipid | 1000 mg/dL |
| Levodopa | 2 mg/dL |
| Metformin | 4 mg/dL |
| Methyldopa | 2 mg/dL |
| Metronidazole | 20 mg/dL |
| Phenylbutazone | 40 mg/dL |
| Rheumatoid factor | 780 000 IU/L |
| Rifampicin | 6.4 mg/dL |
| Salicylic acid | 59 mg/dL |
| Theophylline | 10 mg/dL |
| Total protein (human serum albumin added and native total | 150 g/L |
Potential interference from hemolysis was evaluated using two $\mathrm{K}_2$ -EDTA venous whole blood samples at $6.5\%$ and $\sim 9.5\%$ HbA1c. Test samples were spiked with hemolysates generated by a freeze/thaw cycle of native samples and compared to control samples containing no hemolysate. The sponsor defined significant interference as greater than or equal to $7\%$ difference from the expected
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concentration. The results support the sponsor's claim that hemolysis up to $13.8\%$ does not interfere with their device. The labeling states that "hemolyzed samples with $>14\%$ hemolysis $(2000\mathrm{mg / dL})$ may return an information code."
# ii. Cross reactivity with Hemoglobin Derivatives
Potential cross reactivity with acetylated hemoglobin, carbamylated hemoglobin, labile HbA1c, and glycated albumin was evaluated. Two $\mathrm{K}_2$ -EDTA venous whole blood samples at $6.5\%$ and $\sim 8.5\%$ HbA1c were spiked with the potential interferent (6.0 mg/mL acetylated hemoglobin, 14.1 mg/mL carbamylated hemoglobin, 12.4 mg/mL labile HbA1c, 7.7 mg/mL glycated albumin). Ten replicates of test and control samples were analyzed. The sponsor defined significant interference as greater than or equal to $7\%$ difference from the expected concentration. The testing results support the sponsor's claim in the labeling that there is no cross reactivity with acetylated hemoglobin (4.6 mg/mL), carbamylated hemoglobin (13.8 mg/mL), labile HbA1c (11.4 mg/mL), or glycated albumin (7.7 mg/mL).
Potential cross reactivity from HbA0, HbA1a and HbA1b was evaluated using 100 samples. Two HbA1c concentrations of pooled $\mathrm{K}_2$ -EDTA venous whole blood samples at $6.0\%$ and $9.0\%$ HbA1c (range $4.3\%$ to $13.93\%$ HbA1c) containing HbA0 ( $82\%$ to $95\%$ ), HbA1a ( $0.4\%$ to $1.4\%$ ) and HbA1b ( $0.4\%$ to $2.3\%$ ) were tested in singlicate using an Afinion AS100 analyzer and compared to measurements made using a NGSP comparator method (Tosoh G8 HPLC). The sponsor defined significant interference as greater than or equal to $7\%$ difference from the expected concentration. The testing results support the sponsor's claim in the labeling that there is no cross reactivity with HbA0 (up to $95\%$ ), HbA1a (up to $1.4\%$ ), and HbA1b (up to $2.3\%$ ).
# iii) Hemoglobin Variant Interference
For the hemoglobin variant interference study was performed using a total of 221 $\mathrm{K}_2$ -EDTA venous whole blood samples known to contain common hemoglobin variants (HbA2, HbS, HbC, HbE, HbD, and HbF). Results obtained by a FDA cleared method that has been demonstrated to be free from the hemoglobin interference being tested (Premier Hb9210 for HbA2, HbAS, HbE, and Tosoh Automated Glycohemoglobin Analyzer HLC-723 G8 for HbF). The sponsor's definition of non-significant interference is $\leq 7\%$ mean relative deviation between the Afinion HbA1c Dx result and the NGSP comparator method.
Samples used for interference evaluation:
| Hemoglobin Variant | Number of Samples Tested | % Content of Variant in sample | Range in Concentration in % HbA1c |
| --- | --- | --- | --- |
| HbA2 | 26 | 3.9-5.7 | 5.8-10.6 |
| HbS | 21 | 35-42 | 5.6-8.8 |
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| Hemoglobin Variant | Number of Samples Tested | % Content of Variant in sample | Range in Concentration in % HbA1c |
| --- | --- | --- | --- |
| HbC | 25 | 30-36 | 5.5-9.7 |
| HbE | 20 | 17-26 | 6.1-8.9 |
| HbD | 21 | 27-42 | 6.1-9.4 |
| HbF | 121 | 3.4-28.1 | 5.0-11.3 |
Hemoglobin Variant Result Summary:
| | Mean % Bias (range of relative bias) from Comparator Method | |
| --- | --- | --- |
| Hb Variant | ~6.5 % HbA1c | ~8.5 % HbA1c |
| HbA2 | -3.4 % (-6.2% to -1.4%) | -2.5 % (-6.6% to 2.8%) |
| HbS | -4.1 % (-6.6% to 3.2%) | -1.0 % (-9.9% to 3.5%) |
| HbC | -5.5 % (-8.5% to -1.8%) | -1.8 % (-5.2% to 1.1%) |
| HbE | 3.5 % (1.4% to 7.9%) | 3.7 % (0% to 4.7) |
| HbD | -2.2 % (-4.1% to 0%) | -2.9 % (-5.9% to 0%) |
| HbF | 10.4% HbF is the highest HbF concentration where no significant interference is observed with significant interference defined as > ±7%. | |
The testing results show there is no significant interference for Hemoglobin A2 (≥ 5.7%), Hemoglobin S (≥ 42%), Hemoglobin C (≥ 36%), Hemoglobin E (≥ 26%), and Hemoglobin D (≥ 42%).
The package insert labeling states:
No significant interference for Hemoglobin A2, Hemoglobin C, Hemoglobin D, Hemoglobin E, and Hemoglobin S up to the levels stated.
The results show that there is significant interference due to the presence of HbF at a HbF concentration of > 10.4%. The device labeling contains the following prominent boxed warning:
"This device has significant negative interference with fetal hemoglobin (HbF). HbA1c results are invalid for patients with abnormal amounts of HbF including those with known Heredity Persistence of Fetal Hemoglobin. Refer to the Analytical specificity and Limitations sections in this package insert for details."
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f. Assay cut-off:
Not applicable.
# 2. Comparison studies:
a. Method comparison with predicate device:
A method comparison study was conducted using 120 capillary (fingerstick) whole blood samples and matched $\mathrm{K}_2$ -EDTA venous whole blood samples ranging from 4.6 to $11.4\%$ HbA1c. All samples were tested with two Afinion HbA1c Dx reagent lots at three moderate complexity point-of-care laboratories. The distribution of samples spanned a concentration around the clinical decision points as follows:
| Hemoglobin A1c level | Number of samples | Percent of samples |
| --- | --- | --- |
| 4 – 5% | 5 | 4.2% |
| 5.1 – 6% | 15 | 12.5% |
| 6.1 – 6.5% | 30 | 25.0% |
| 6.6 – 7% | 30 | 25.0% |
| 7.1 – 8% | 20 | 16.7% |
| 8.1 – 9% | 10 | 8.3% |
| 9.1 – 15% | 10 | 8.3% |
| Total samples | 120 | 100% |
Singlicate capillary and venous whole blood results from the Afinion HbA1c Dx test system were compared to average results from matched $\mathrm{K}_2$ -EDTA venous whole blood samples tested in duplicate using a NGSP comparator method (Tosoh G8 HPLC).
Deming (weighted) and Passing-Bablok regression analyses were performed for the Afinion HbA1c Dx assay versus the comparator method.
Fingerstick capillary whole blood samples
Weighted Deming regression:
| y-intercept | Slope | 95% CI |
| --- | --- | --- |
| 0.000 | 0.997 | 0.966 – 1.027 (slope) -0.209 – 0.201 (intercept) |
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Passing-Bablok regression:
| y-intercept | Slope | 95% CI |
| --- | --- | --- |
| -0.040 | 1.000 | 0.971 – 1.033 (slope) -0.271 – 0.165 (intercept) |
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The following biases for fingerstick blood samples were observed between the Afinion HbA1c Dx and the comparator method:
| Decision Level | Weighted Deming regression | | Passing-Bablok regression | |
| --- | --- | --- | --- | --- |
| | Bias | % Bias | Bias | % Bias |
| 5.0% | -0.017 | -0.335 | -0.040 | -0.800 |
| 6.5% | -0.022 | -0.334 | -0.040 | -0.615 |
| 8.0% | -0.027 | -0.334 | -0.040 | -0.500 |
| 12.0% | -0.040 | -0.333 | -0.040 | -0.333 |
# Venous whole blood samples
Weighted Deming regression:
| y-intercept | Slope | 95% CI |
| --- | --- | --- |
| 0.053 | 0.991 | 0.959 – 1.022 (slope) -0.162 – 0.270 (intercept) |
Passing-Bablok regression:
| y-intercept | Slope | 95% CI |
| --- | --- | --- |
| -0.030 | 1.000 | 0.968 – 1.031 (slope) -0.232 – 0.184 (intercept) |
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The following biases were observed between the Afinion HbA1c Dx and the comparator method:
| Decision Level | Weighted Deming regression | | Passing-Bablok regression | |
| --- | --- | --- | --- | --- |
| | Bias | % Bias | Bias | % Bias |
| 5.0% | 0.010 | 0.195 | -0.030 | -0.600 |
| 6.5% | -0.003 | -0.052 | -0.030 | -0.462 |
| 8.0% | -0.017 | -0.206 | -0.030 | -0.375 |
| 12.0% | -0.051 | -0.429 | -0.030 | -0.250 |
# Total Error Calculations
Using the results of bias estimation (\% Bias) in the method comparison study and precision estimates, Total Error (TE) at four concentrations (5.0%, 6.5%, 8.0%, and 12.0%) was calculated using all components of variance as stated in the special controls as follows: $\% \mathrm{TE} = |\% \mathrm{Bias}| + 1.96 * \% \mathrm{CV}^{*}(1 + \% \mathrm{Bias} / 100)$ . The results are presented in the tables below.
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Capillary fingerstick samples:
| Level | %CV | Weighted Deming regression | | Passing-Bablok regression | |
| --- | --- | --- | --- | --- | --- |
| | | %Bias | %TE | %Bias | %TE |
| 5.0% | 2.03 | -0.335 | 4.30 | -0.800 | 4.75 |
| 6.5% | 1.58 | -0.334 | 3.42 | -0.615 | 3.69 |
| 8.0% | 1.49 | -0.334 | 3.24 | -0.500 | 3.40 |
| 12.0% | 1.30 | -0.333 | 2.87 | -0.333 | 2.87 |
EDTA venous whole blood samples (based on external point-of-care precision study):
| Level | %CV | Weighted Deming regression | | Passing-Bablok regression | |
| --- | --- | --- | --- | --- | --- |
| | | %Bias | %TE | %Bias | %TE |
| 5.0% | 1.78 | 0.195 | 3.69 | -0.600 | 4.07 |
| 6.5% | 1.48 | -0.052 | 2.95 | -0.462 | 3.35 |
| 8.0% | 1.36 | -0.206 | 2.87 | -0.375 | 3.03 |
| 12.0% | 1.22 | -0.429 | 2.81 | -0.250 | 2.64 |
EDTA venous whole blood samples (based on internal precision study):
| Level | %CV | Weighted Deming regression | | Passing-Bablok regression | |
| --- | --- | --- | --- | --- | --- |
| | | %Bias | %TE | %Bias | %TE |
| 5.0% | 1.64 | 0.195 | 3.42 | -0.600 | 3.80 |
| 6.5% | 1.51 | -0.052 | 3.01 | -0.462 | 3.41 |
| 8.0% | 1.31 | -0.206 | 2.77 | -0.375 | 2.93 |
| 12.0% | 1.42 | -0.429 | 3.20 | -0.250 | 3.03 |
b. Matrix comparison:
Not applicable. Venous $\mathrm{K}_2$ -EDTA and capillary are the only matrices claimed for this assay.
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):
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Not applicable.
4. Clinical cut-off:
See expected values, below.
5. Expected values/Reference range:
The diagnostic cut-off is 6.5 % HbA1c. Patients with HbA1c values in the range 5.7-6.4 % are identified as having an increased risk for developing diabetes¹,².
¹International Expert Committee, International Expert Committee Report on the Role of the A1c Assay in the Diagnosis of Diabetes. Diabetes Care 2009; 32(7):1237-1334.
²American Diabetes Association. Classification and Diagnosis of Diabetes. Diabetes Care 2018; 41(Suppl. 1): S13-S27.
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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