MINICAP HB A1C, MINICAP FLEX-PIERCING, HB A1C CAPILLARYS CONTROLS, HBA1C CAPILLARYS CALIBRATORS

K133344 · Sebia · LCP · Mar 28, 2014 · Hematology

Device Facts

• Record ID: K133344
• Device Name: MINICAP HB A1C, MINICAP FLEX-PIERCING, HB A1C CAPILLARYS CONTROLS, HBA1C CAPILLARYS CALIBRATORS
• Applicant: Sebia
• Product Code: LCP · Hematology
• Decision Date: Mar 28, 2014
• Decision: SESE
• Submission Type: Traditional
• Regulation: 21 CFR 864.7470
• Device Class: Class 2

Intended Use

The MINICAP HbA1c kit is designed for separation and quantification of the HbA1c glycated fraction of hemoglobin in human whole blood, by capillary electrophoresis in alkaline buffer with the MINICAP FLEX-PIERCING instrument. Measurement of hemoglobin A1c is effective in monitoring long-term glycemic control in individuals with diabetes mellitus. Results are provided in IFCC (mmol/mol) and NGSP (%Hb A1c) units. The MINICAP Hb A1c kit is designed for Professional Use Only. For In Vitro Use. The Hb A1c CAPILLARY Controls are designed for the quality control of human glycated hemoglobin A1c quantification with SEBIA capillary electrophoresis procedures using the MINICAP HbA1c kit with the MINICAP FLEX-PIERCING automated instrument. The Hb A1c CAPILLARY Controls are designed for Professional Use Only. For In Vitro Diagnostic Use. The HbA1c CAPILLARY Calibrators are designed for the calibration and migration control of human glycated hemoglobin A1c quantification with SEBIA capillary electrophoresis procedures using the MINICAP HbA1c kit with the MINICAP FLEX-PIERCING automated instrument. The HbA1c CAPILLARY Calibrators are designed for Professional Use Only. For In Vitro Diagnostic Use.

Device Story

MINICAP FLEX-PIERCING is an automated analyzer for HbA1c quantification in human whole blood (K2EDTA anticoagulant). System uses capillary electrophoresis in alkaline buffer; separates hemoglobin fractions based on electrophoretic mobility; detects hemoglobins via absorbance at 415 nm. Instrument features 2 parallel silica capillaries; performs automated sample dilution and hemolysis. Used in clinical laboratories by trained personnel. Output includes HbA1c fraction quantification in IFCC (mmol/mol) and NGSP (%) units. High resolution allows quantification in presence of variants (HbS, HbC, HbD, HbE, HbF) and common interferents. Results assist clinicians in monitoring glycemic control in diabetic patients.

Clinical Evidence

Bench testing only. Precision/reproducibility evaluated per CLSI EP5-A2; total CVs ranged 0.0-4.0%. Linearity confirmed across 4.8-13.8% HbA1c range. Interference studies showed no significant impact (≤0.3% HbA1c) from triglycerides, bilirubin, ascorbic acid, urea, rheumatoid factor, glybenclamide, carbamylated Hb, labile HbA1c, acetylated Hb, or variants (HbS, E, D, C, F). Method comparison (n=367 samples across internal/external studies) showed high correlation (r=0.998) with predicate.

Technological Characteristics

Capillary electrophoresis in free solution; alkaline buffer; silica capillaries; 415 nm absorbance detection; deuterium lamp and LED light source. Automated sample dilution/hemolysis. Standalone instrument with PC interface. Software-controlled. Standardized to NGSP/IFCC. Dimensions: 44cm x 41.5cm x 58cm; Weight: 32kg.

Indications for Use

Indicated for the separation and quantification of HbA1c in human whole blood to monitor long-term glycemic control in diabetic patients. For professional in vitro diagnostic use.

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

• CAPILLARYS Hb A1c kit (k122101)
• CAPILLARYS 2 FLEX-PIERCING instrument (k122101)
• Hb A1c CAPILLARY Controls (k122101)
• Hb A1c CAPILLARY Calibrators (k122101)

Submission Summary (Full Text)

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K133344
MAR 2 8 2014

### 510(K) Summary

SEBIA's manufacturing and corporate office is located at:

Parc Technologique Léonard de Vinci, Rue Léonard de Vinci CP 8010 LISSES 91008 EVRY Cedex, FRANCE

Phone: [phone]; Fax: [phone]

In the United States, the product will be distributed by:

SEBIA Inc. Suite 400 - 1705 Corporate drive NORCROSS GA 30093, USA Phone 770 446 - 3707; Fax 770 446 - 8511 Contact person: Karen Anderson Prepared on March 26, 2014

- Product/Device Names: MINICAP Hb A1c (PN 2215), MINICAP FLEX-PIERCING (PN 1232), Hb A1c CAPILLARY Calibrators (PN 4755), Hb A1c CAPILLARY Controls (PN 4774)
Common Name: glycosylated hemoglobin

Product Regulation Name: glycosylated hemoglobin assay

The MINICAP Hb A1c type of devices/assays are classified by FDA as Class II, under Regulation No. 21 CFR 864.7470. SEBIA is seeking clearance to import the assay described above, and by this submission is notifying FDA of its intent to market these products in the United States.

| Product Code | Classification | Regulation Section | Panel |
|--------------|-------------------|---------------------------------------------------------------------------|-----------------|
| LCP | II | 21 CFR 864.7470<br>Glycosylated<br>hemoglobin assay | Hematology (81) |
| JIS | II | 21 CFR 862.1150<br>Calibrator | Chemistry (75) |
| JJX | Class I, Reserved | 21 CFR 862.1660<br>Quality control<br>material (assayed<br>and unassayed) | Chemistry (75) |

Product Nomenclature:

### HEMOGLOBINS A1C BY CAPILLARY ELECTROPHORESIS

Establishment Registration Number:

8023024

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STANDARDS: MINICAP Hb A1c test is standardized according to NGSP and IFCC requirements/quidelines.

This submission is limited to the MINICAP Hb A1c kit (PN 2215) using the MINICAP FLEX-PIERCING instrument (PN 1232) and the performance using the Hb A1c CAPILLARY Controls (PN 4774) with the system and Hb A1c CAPILLARY Calibrators (PN 4755)

The SEBIA Hb A1c CAPILLARY Controls (PN 4774) and Hb A1c CAPILLARY Calibrators (PN 4755) were FDA cleared (K122101), December 6, 2012.

### Substantial Equivalence to Predicate Devices:

For the separation and quantification of the Hb A1c glycated fraction of hemoglobin in human blood, by capillary electrophoresis in an alkaline buffer using the MINICAP Hb A1c kit with the MINICAP FLEX-PIERCING instrument.

The new device, MINICAP Hb A1c procedure using the MINICAP FLEX-PIERCING instrument, utilizes the previous cleared SEBIA Hb A1c CAPILLARY Calibrators and Hb A1c CAPILLARY Controls (K122101).

The performance of the MINICAP Hb A1c kit using the MINICAP FLEX-PIERCING instrument. Hb A1c CAPILLARY Calibrators and Hb A1c CAPILLARY Controls was compared to the predicate device, CAPILLARYS Hb A1c kit using the CAPILLARYS 2 FLEX-PIERCING instrument, the Hb A1c CAPILLARY Calibrators and the Hb A1c CAPILLARY Controls (K122101).

Both the new device (MINICAP Hb A1c kit & MINICAP FLEX-PIERCING instrument using the Hb A1c CAPILLARY Calibrators and the Hb A1c CAPILLARY Controls) and the predicate device (CAPILLARYS Hb A1c kit & CAPILLARYS 2 FLEX-PIERCING instrument using the Hb A1c CAPILLARY Calibrators and the Hb A1c CAPILLARY Controls) use capillary electrophoresis technology. The devices compared were found to be substantially equivalent in function, concept, principle, technique, use, safety and effectiveness,

The 510(K) number of the predicate device, CAPILLARYS Hb A1c using CAPILLARYS 2 FLEX-PIERCING instrument, the Hb A1c CAPILLARY Calibrators and the Hb A1c CAPILLARY Controls predicate device, was FDA cleared as K122101 on December 6, 2012.

| 510K Table of Predicate Devices | | |
|-------------------------------------------------|---------|------------------|
| Predicate Device | 510K | Clearance Date |
| CAPILLARYS Hb A1c<br>CAPILLARYS 2 FLEX PIERCING | K122101 | December 6, 2012 |
| Hb A1c CAPILLARY Controls | K122101 | December 6, 2012 |
| Hb A1c CAPILLARY Calibrators | K122101 | December 6, 2012 |

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# DEVICE DESCRIPTION

The MINICAP FLEX-PIERCING instrument is an automated analyzer which performs a complete hemoglobin profile for the quantitative analysis of HbA1c fraction. The hemoglobins, separated in silica capillaries, are directly detected by their absorbance at 415 nm. The assay is performed on the hemolysate of whole blood samples collected in tubes containing K2EDTA as anticoagulant.

Quantitative determination of hemoglobin A1c is effective in monitoring middle-term blood glucose control in diabetic individuals.

The MINICAP Hb A1c procedure performed with the MINICAP FLEX-PIERCING instrument has been certified by the National Glycohemoglobin Standardization Program (NGSP).

Electrophoresis is a well established technique routinely used in clinical laboratories for measuring components from body fluids, including HbA1c glycated fraction. The MINICAP FLEX-PIERCING instrument has been developed to provide complete automation of this testing with fast separation and good resolution. In many aspects, the methodology can be considered as an intermediary type of technique between classical zone electrophoresis and liquid chromatography.

The MINICAP FLEX-PIERCING instrument uses the principle of capillary electrophoresis in free solution. With this technique, charged molecules are separated by their electrophoretic mobility in an alkaline buffer with a specific pH. Separation also occurs according to the electrolyte pH and electroosmotic flow.

The MINICAP FLEX-PIERCING instrument has silica capillaries functioning in parallel allowing 2 simultaneous analyses for HbA1c quantification from whole blood sample. A sample dilution with hemolysing solution is prepared and injected by aspiration at the anodic end of the capillary. A high voltage protein separation is then performed and direct detection of the hemoglobins is made at the cathodic end of the capillary at 415 nm, which is the absorbance wave length specific to hemoglobins. Before each run, the capillaries are washed with a wash solution and prepared for the next analvsis with buffer.

Direct detection provides accurate relative quantification of individual hemoglobin Are fraction. In addition, the high resolution of MINICAP Hb A1c procedure allows the quantification of HbA1c, even in the presence of labile HbArc, carbamylated and acetylated hemoglobins, and major hemoglobin variants such as HbS, HbC, HbD, HbE and HbF and common interfering factors such as Triglycerides, Bilirubin, Ascorbic Acid, Urea, Rheumatoid factor and Glybenclamide as outline in the package insert labeling.

By using alkaline pH buffer, normal and abnormal (or variant) hemoglobins are detected in the following order, from cathode to anode: A2/C, E, S/D, F, A0, other Hb (including minor Hb A1) and then A1c.

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### INTENDED USE

The MINICAP Hb A1c kit is designed for separation and quantification of the HbA1c glycated fraction of hemoglobin in human whole blood, by capillary electrophoresis in alkaline buffer with the MINICAP FLEX-PIERCING instrument. Measurement of hemoglobin A1c is effective in monitoring long-term glycemic control in individuals with diabetes mellitus. Results are provided in IFCC (mmol/mol) and NGSP (%Hb A1c) units.

The MINICAP Hb A1c kit is designed for Professional Use Only.

The Hb A1c CAPILLARY Controls are designed for the quality control of human glycated hemoglobin A1c quantification with SEBIA capillary electrophoresis procedures using the MINICAP HbA1c kit with the MINICAP FLEX-PIERCING automated instrument.

The Hb A1c CAPILLARY Controls are designed for Professional Use Only.

The Hb A1c CAPILLARY Calibrators are designed for the calibration and migration control of human glycated hemoglobin A1c quantification with SEBIA capillary electrophoresis procedures using the MINICAP HbA1c kit with the MINICAP FLEX-PIERCING automated instrument.

The Hb A1c CAPILLARY Calibrators are designed for Professional Use Only.

For In Vitro Diagnostic Use.

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## PRODUCT DESCRIPTION

## 1. MINICAP FLEX PIERCING instrument, Part Number 1232

### 2. Reagent Kit

The MINICAP Hb A1c kits, Hb A1c CAPILLARY Controls and Hb A1c CAPILLARY Calibrators are used with the MINICAP FLEX- PIERCING system.

The configurations of the components are summarized:

- . MINICAP Hb A1c kits in Table I.
- . Hb A1c CAPILLARY Calibrators in Table II.
- Hb A1c CAPILLARY Controls in Table III. .
- Reagents that are required to perform the test but are sold separately in Table IV. .

For additional details, see Package Inserts and instrument operators manual. Each kit, control and calibrators is supplied with a Package Insert which contains instruction for use and all the necessary information on the reagents needed to run the tests. Each Package Insert also contains information on storage conditions, shelf life and signs of deterioration of the components and the reagents sold separately.

| ITEMS | PN 2215 |
|-------------------------------------|----------------------|
| Buffer (ready to use) | 2 vials, 250 mL each |
| Hemolysing solution (ready to use) | 1 vial, 225 mL |
| Wash solution (stock solution) | 1 vial, 25 mL |
| Reagent Cups | 1 pack of 125 |
| Filters | 3 filters |
| Bins for used cups | 4 bins |
| Hemolysing solution bar code labels | 5 sheets of 4 labels |

# TABLE I. REAGENTS AND MATERIALS SUPPLIED IN THE MINICAP Hb A1c KIT (PN 2215)

### TABLE II. REAGENTS AND MATERIALS SUPPLIED WITH Hb A1c CAPILLARY CALIBRATORS (PN 4755)

| ITEMS | PN 4755 |
|---------------------------------------------|----------------------------|
| Hb A1c CAPILLARY Calibrator 1 (green cap) | 1 vial of each, 600µL each |
| Hb A1c CAPILLARY Calibrator 2 (red cap) | |
| Barcode label Hb A1c CAPILLARY Calibrator 1 | 1 |
| Barcode label Hb A1c CAPILLARY Calibrator 2 | 1 |

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TABLE III. REAGENTS AND MATERIALS SUPPLIED WITH Hb A1c CAPILLARY CONTROLS (PN 4774)

| ITEMS | PN 4744 |
|------------------------------------------|----------------------------|
| Hb A1c CAPILLARY Control 1 ( white cap ) | 1 vial of each, 600µL each |
| Hb A1c CAPILLARY Control 2 ( black cap) | |
| Barcode label HbA1c CAPILLARY Control 1 | 2 |
| Barcode label HbA1c CAPILLARY Control 2 | 2 |
| White dilution segments* | 4 |
| Grey dilution segments* | 4 |

* Not used with the MINICAP FLEX-PIERCING instrument

# TABLE IV. REAGENTS AND MATERIALS REQUIRED BUT NOT SUPPLIED IN THE MINICAP HbA1c KIT, Hb A1c CAPILLARY CONTROLS OR Hb A1c CAPILLARY CALIBRATORS

| ITEMS | PN | COMPONENTS |
|------------------------------------------------|------------|--------------------------|
| CAPICLEAN | 2058 | 1 vial, 25 mL |
| CAPILLARYS / MINICAP Wash Solution | 2052 | 2 vials, 75 mL |
| Tubes and caps for controls | 9202, 9205 | 200 per box, 500 per box |
| MINICAP Reagent Cups | 2280 | 250 per box |
| Lids for bins for used reagent cups | 2286 | 12 per box |
| "AUTOMATIC LOW VOLUME" bar code labels | 9208 | 20 per box |
| "MANUAL LOW VOLUME" bar code labels | 9209 | 20 per box |
| MINICAP FLEX-PIERCING centering rings | 1612 | 27 per box |
| PHORESIS software | 1110 | |
| MINICAP | 1232 | |
| FLEX-PIERCING INSTRUMENT | | |
| Update HbA1c kit for MINICAP FLEX-<br>PIERCING | 1238 | |

### LABELING

Labeling contained in this submission includes:

- A. MINICAP Hb A1c operators manual
- B. MINICAP Hb A1c package insert
- C. Hb A1c CAPILLARY Controls package insert
- D. Hb A1c CAPILLARY Calibrators package insert

and the box labels and the product labels of the MINICAP Hb A1c kit, MINICAP Hb A1c CAPILLARY Controls, Hb A1c CAPILLARY Calibrators and of the reagents and materials required but not supplied.

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## STUDY SUMMARY

- 1. Analytical performance:
- a. Precision/Reproducibility:

The reproducibility studies have been performed according to CLSI Guideline "EPS-A2: Evaluation of Precision Performance of Clinical Chemistry Devices".

### Reproducibility between lots and instruments

Eight (8) different blood samples were run using the MINICAP Hb A1c procedure in both capillaries of 3 different MINICAP FLEX-PIERCING instruments and with 3 lots of MINICAP Hb A1c kits. The analyzed blood samples included 3 samples with normal HbA% level (No. 1, 2 and 3), 1 sample with HbArelevel close to the cut-off value (No. 4) and 4 samples with elevated HbAre level (No. 5, 6, 7 and 8). In this study, each blood sample was analyzed on both capillaries from each instrument, including 60 runs over 10 working days (at 2 different times of the day). Within each run, samples were analyzed in duplicate.

The following tables summarize the within-run and total instrument-reagent C.V. % ranges for the HbA1c concentrations (in mmol/mol) and percentages.

| | Within-run reproducibility | | | Total reproducibility | | | Mean (HbA1c<br>concentration<br>- mmol/mol) | Within-run reproducibility | | Total reproducibility | |
|---------------|----------------------------|------------|------------|-----------------------|------------------|---------------|---------------------------------------------|----------------------------|------------|-----------------------|------------------|
| | Mean<br>(% HbA1c) | CV min (%) | CV max (%) | Total CV min (%) | Total CV max (%) | | | CV min (%) | CV max (%) | Total CV min (%) | Total CV max (%) |
| Sample No. 1 | 5.2 | 0.9 | 2.0 | 0.9 | 2.2 | Sample No. 1 | 33 | 0.9 | 3.5 | 0.9 | 3.5 |
| Sample No. 2 | 5.4 | 0.9 | 2.1 | 0.9 | 2.1 | Sample No. 2 | 36 | 1.1 | 4.0 | 1.2 | 4.0 |
| Sample No. 3 | 5.5 | 0.5 | 2.0 | 0.7 | 2.1 | Sample No. 3 | 37 | 0.0 | 3.0 | 1.4 | 3.3 |
| Sample No. 4 | 6.4 | 0.5 | 1.9 | 0.8 | 1.9 | Sample No. 4 | 47 | 1.2 | 2.6 | 1.3 | 2.6 |
| Sample No. 5 | 7.9 | 0.7 | 1.4 | 0.8 | 1.6 | Sample No. 5 | 63 | 0.7 | 1.6 | 0.7 | 2.2 |
| Sample No. 6 | 9.1 | 0.0 | 1.1 | 0.0 | 1.1 | Sample No. 6 | 76 | 0.0 | 1.4 | 0.0 | 1.4 |
| Sample No. 7 | 10.1 | 0.6 | 1.1 | 0.6 | 1.2 | Sample No. 7 | 87 | 0.3 | 1.3 | 0.3 | 1.4 |
| Sample No. 8 | 12.3 | 0.4 | 1.2 | 0.6 | 1.8 | Sample No. 8 | 110 | 0.4 | 1.4 | 0.6 | 2.2 |
| CV (%) ranges | | 0.0 | 2.1 | 0.0 | 2.2 | CV (%) ranges | | 0.0 | 4.0 | 0.0 | 4.0 |

### Reproducibility within the same capillary and between capillaries from the same instrument

Eight (8) different blood samples were run using the MINICAP Hb A1c procedure in both capillaries of the same MINICAP FLEX-PIERCING instrument and with 1 lot of MINICAP Hb A1c kit. The analyzed blood samples included 3 samples with normal HbAic level (No. 1, 2 and 3), 1 sample with HbA . level close to the cut-off value (No. 4) and 4 samples with elevated HbA% . level (No. 5, 6, 7 and 8). In this study, each blood sample was analyzed on both capillaries from the same

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instrument, including 40 runs over 20 working days (at 2 different times of the day). Within each run, samples were analyzed in duplicate.

The results for HbAje concentrations (in mmol/mol) and percentages are summarized in the following tables.

For reproducibility within the same capillary, maximal CV's have been calculated for each blood sample from pooled data obtained on each capillary.

| | Sample<br>No. 1 | Sample<br>No. 2 | Sample<br>No. 3 | Sample<br>No. 4 | Sample<br>No. 5 | Sample<br>No. 6 | Sample<br>No. 7 | Sample<br>No. 8 |
|-----------------------------------------------|-----------------|-----------------|-----------------|-----------------|-----------------|-----------------|-----------------|-----------------|
| Mean (% HbA1c) | 5.2 | 5.2 | 5.7 | 6.4 | 7.8 | 9.0 | 10.1 | 11.9 |
| Within-run<br>reproducibility<br>(CV %) | 1.0 | 1.1 | 1.0 | 0.9 | 1.1 | 0.9 | 0.7 | 1.1 |
| Within-capillary<br>reproducibility<br>(CV %) | 1.4 | 1.4 | 1.5 | 1.1 | 0.7 | 0.8 | 0.8 | 0.9 |
| Between-run<br>reproducibility<br>(CV %) | 0.0 | 0.3 | 0.7 | 0.0 | 0.0 | 0.0 | 0.3 | 0.0 |
| Between-day<br>reproducibility<br>(CV %) | 0.7 | 0.0 | 0.5 | 0.6 | 0.2 | 0.4 | 0.3 | 0.1 |
| Total (CV%) | 1.2 | 1.1 | 1.3 | 1.1 | 1.1 | 1.0 | 0.8 | 1.1 |

8

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| | Sample<br>No. 1 | Sample<br>No. 2 | Sample<br>No. 3 | Sample<br>No. 4 | Sample<br>No. 5 | Sample<br>No. 6 | Sample<br>No. 7 | Sample<br>No. 8 |
|-----------------------------------------------|-----------------|-----------------|-----------------|-----------------|-----------------|-----------------|-----------------|-----------------|
| Mean (HbA1c<br>concentration –<br>mmol/mol) | 33 | 34 | 38 | 46 | 62 | 75 | 87 | 106 |
| Within-run<br>reproducibility<br>(CV %) | 1.7 | 1.9 | 1.7 | 1.1 | 0.8 | 1.2 | 0.8 | 1.2 |
| Within-capillary<br>reproducibility<br>(CV %) | 2.5 | 2.8 | 2.4 | 1.3 | 0.8 | 1.2 | 1.0 | 0.9 |
| Between-run<br>reproducibility<br>(CV %) | 0.0 | 1.4 | 0.3 | 0.5 | 0.0 | 0.0 | 0.4 | 0.0 |
| Between-day<br>reproducibility<br>(CV %) | 1.3 | 0.0 | 0.6 | 0.5 | 0.0 | 0.6 | 0.3 | 0.2 |
| Total (CV %) | 2.2 | 2.3 | 1.8 | 1.3 | 0.8 | 1.3 | 1.0 | 1.2 |

# b. Linearity/assay reportable range:

The linearity of the MINICAP HbA1c procedure was evaluated based on CLSI EP6-A quideline "Evaluation of the Linearity Quantitative Measurement Procedures: A Statistical Approach". Two blood samples, including a normal sample with HbA1c concentration at 4.8% (29 mmol/mo)) and an elevated HbA1c level sample with HbA1c concentration at 13.8% (127 mmol/mol) were mixed within different proportions and the dilutions were electrophoresed with the MINICAP HbA1c assay kit using the MINICAP FLEX-PIERCING instrument. Samples were analyzed in duplicate.

A polynomial regression analysis was performed, it allows to conclude on the linearity of MINICAP Hb A1c procedure performed with the MINICAP FLEX-PIERCING instrument for HbA1c fraction within the entire range studied.

HbA1c (%) The 1st order linear regression generated is: Y=0.08982x+4.764, r2=0.998, r=0.999 The linearity range is 4.8 - 13.8% HbA1c

HbA1c (mmol/mol)

The 1st order linear regression generated is:

Y=0.9855x+28.41. r2=0.999. r=0.999

The linearity range is 29 - 127 mmol/mol HbA1c

In addition, 3 different characteristic blood samples, including a normal sample with HbA1c concentration at 5.0 % HbA1c (31 mmol/mol), a sample with HbA1c level close to the cut-off value at 6.3 % HbA1c (46 mmol/mol) and an elevated HbA1c level sample with HbA1c concentration at

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9.3 % HbA1c (79 mmol/mol), were all serially diluted in hemolysing solution and electrophoresed with the MINICAP Hb A1c procedure. The tests were determined to be linear within the entire ranges studied from 2.5 to 31.1 g/dL total hemoglobin and HbA1c fraction concentration and percentage were not affected by the hemoglobin concentration of the samples.

### c. Detection limit:

The Limit of Blank (LoB) and Limit of Detection (LoD) were determined by assaying a five zero samples (blank) and six low HbA1c samples according to CLSI quideline EP17-A . The results are as follows:

LoB= 0.3%, LoD = 1.1%

The claimed measuring range, 4.8- 13.8% (29 - 127 mmol/mol), is based on linearity.

### d. Analytical specificity:

The interference studies have been performed according to the CLSI Guideline "EPT-A2: Interference Testing in Clinical Chemistry".

i) Studies were performed to assess common or known substances that could interfere with the MINICAP HbA1c assay kit. The interfering substances were evaluated in whole blood samples that contained four different concentrations of A1c (~5.0%. ~8.8% and ~11.9%). Samples containing various concentrations of potential interferents were tested and the results compared to those obtained from control samples containing no potential interfering substances. The definition of non-significant interference is ≤ 0,3% HbA1c between the tested and the control samples. The results are as follows:

| Potential interfering substance | Concentration at which no significant<br>interference (≤0.3%) was observed |
|---------------------------------|----------------------------------------------------------------------------|
| Triglycerides | 3.07 g/dL (35.1 mM) |
| Bilirubin | 25.8 mg/dL (442 μM) |
| Ascorbic acid | 60 mg/dL (3.41 mM) |
| Urea | 291 mg/dL (48.5 mM) |
| Rheumatoid factor | 2178 IU/mL |
| Glybenclamide | 3 mg/dL |

ii) To study interference from Carbamlyated hemoglobin, four whole blood patient samples with A1c concentrations at ~5.7%, ~6.9% and ~12.4% were split into two aliguots. One aliguot, at each A1c level, was spiked with 8.11 mg/dL (1 mmol/L) of Potassium Cyanate and incubated for 3 hours at 37°C. Another aliquot, at each A1c level, was incubated for 3 hours at 37°C. Samples were then analyzed on the MINICAP FLEX-PIERCING instrument using the MINICAP HbA1c assay kit, Samples were analyzed in triblicate. The definition of non-significant interference is ≤ 0.3 HbA1c% between the tested and the control samples.

To conclude Carbamylated hemoglobin (≤ 8.7 %) does not interfere with this assay.

iii) To study interference from Labile HbA1c, four whole blood patient samples with A1c concentrations at ~4.7%, ~6.8%, ~8.8% and ~12.7% were split into two aliguots. One aliguot. at each A1c level, was spiked with 1800 mg/dL (0,5 mol/L) of glucose and incubated for 3 hours at 37°C. Another aliquot, at each A1c level, was incubated for 3 hours at 37°C. Samples were then analyzed on the MINICAP FLEX-PIERCING instrument using the MINICAP HbA1c assay kit. Samples were analyzed in triplicate. The definition of non-significant interference is ≤ 0.3 HbA1c% between the tested and the control samples.

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To conclude Labile Hb A1c (≤ 14.8 %) does not interfere with this assay.

iv) To study interference from Labile HbA1c, four whole blood patient samples with A1c concentrations at ~5.2%, ~6.6%, ~9.4% and ~11.7% were split into two aliguots. One aliguot, at each A1c level, was spiked with 180 mg/dL (10 mmol/L) of acetylsalicylic and incubated for 4 hours at 37°C. Another aliquot, at each A1c level, was incubated for 4 hours at 37°C. Samples were then analyzed on the MINICAP FLEX-PIERCING instrument using the MINICAP HbA1c assay kit, Samples were analyzed in triplicate. The definition of non-significant interference is ≤ 0.3 HbA1c% between the tested and the control samples.

To conclude Acetylated hemoglobin (≤ 3.0 %) does not interfere with this assay.

v) A hemoglobin variant interference study was carried out using samples known to contain Hemoglobin variants S, E, D and C. These variant samples were tested on the MINICAP FLEX-PIERCING instrument using the MINICAP HbA1c assay kit. The definition of non-significant interference is ±10% difference between the candidate method and a NGSP reference method (performed in a NGSP laboratory).

The testing results show there is no significant interference for HbS (≤ 40.5%), HbE (≤ 24.7%), HbD (≤ 41.0%) and HbC (≤ 37.0%).

vi) An additional variant interference study was carried out to study the variant interference from Hemoglobin F. 16 whole blood samples with HbA1c concentrations of ~5.3% and ~11.6% contained various concentrations of HbF (2.3 to 19.7%) were tested on the MINICAP FLEX-PIERCING instrument using the MINICAP HbA1c assay kit. The definition of non-significant interference is ±10% difference between the candidate method and a NGSP reference method (performed in a NGSP laboratory).

The testing results show there is no significant interference for HbF ≤ 19.7%.

- 2. Comparison studies:
- a. Method comparison with predicate device:

The correlation studies have been performed according to CLSI Guideline "EP9-A2: Method Comparison and Bias Estimation Using Patient Samples".

### Internal Study :

101 whole blood samples with HbA1c ranging from 4.8% (29 mmol/mol) to 13.3% (122 mmol/mol) were analyzed in singlicate using the MINICAP HbA1c assay kit on the MINICAP FLEX-PIERCING instrument (candidate device) and on the CAPILLARYS Hb A1c assay on the CAPILLARYS 2 FLEX-PIERCING instrument. The linear regression correlation was calculated as follows:

| HbA1c | Correlation coefficient | y-Intercept | Slope | Range of values<br>MINICAP Hb A1c |
|-----------------------------|-------------------------|-------------|-------|-----------------------------------|
| Percentage<br>(%) | 0.998 | 0.165 | 0.982 | 4.8 - 13.3 |
| Concentration<br>(mmol/mol) | 0.998 | 1.262 | 0.985 | 29 - 122 |

### External study No.1

126 whole blood samples with HbA1c ranaing from 4.8% (29 mmol/mol) to 13.6% (125 mmo//mol) were analyzed in singlicate using the MINICAP HbA1c assay kit on the MINICAP FLEX-PIERCING

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instrument (candidate device) and on the CAPILLARYS Hb A1c assay on the CAPILLARYS 2 FLEX-PIERCING instrument. The linear regression correlation was calculated as follows:

| HbA1c | Correlation coefficient | y-Intercept | Slope | Range of values<br>MINICAP Hb A1c |
|-----------------------------|-------------------------|-------------|-------|-----------------------------------|
| Percentage<br>(%) | 0.998 | - 0.032 | 0.997 | 4.8 - 13.6 |
| Concentration<br>(mmol/mol) | 0.998 | - 0.396 | 0.996 | 29 - 125 |

### External study No. 2

140 whole blood samples with HbA1c ranging from 4.8% (29 mmol/mol) to 13.1% (119 mmol/mol) were analyzed in singlicate using the MINICAP HbA1c assay kit on the MINICAP FLEX-PIERCING instrument (candidate device) and on the CAPILLARYS Hb A1c assay on the CAPILLARYS 2 FLEX-PIERCING instrument. The linear regression correlation was calculated as follows:

| HbA₁c | Correlation coefficient | y-Intercept | Slope | Range of values<br>MINICAP Hb A1c |
|-----------------------------|-------------------------|-------------|-------|-----------------------------------|
| Percentage<br>(%) | 0.998 | - 0.057 | 1.019 | 4.8 - 13.1 |
| Concentration<br>(mmol/mol) | 0.998 | - 0.316 | 1.023 | 29 - 119 |

### b. Matrix comparison:

A total of 41 random matched sample pairs (K2 EDTA and K3 EDTA) were tested on the MINICAP FLEX-PIERCING instrument using the MINICAP HbA1c assay kit. The linear regression is presented in the table below:

| Fraction | Number of<br>samples | Correlation<br>coefficient | y-intercept | Slope | Range of<br>HbA1c<br>fractions<br>(test) |
|---------------------|----------------------|----------------------------|-------------|-------|------------------------------------------|
| HbA1c (%) | 41 | 0,999 | 0,039 | 0,998 | 4,9 - 13,3 |
| HbA1c<br>(mmol/mol) | 41 | 0,999 | 0,091 | 1,001 | 30 - 122 |

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### SUBSTANTIAL EQUIVALENCE

The performance and comparative studies of the MINICAP Hb A1c test with the MINICAP FLEX-PIERCING instrument were performed using SEBIA's commercially available materials and standard procedures.

In the comparative studies, commercially available materials and standard procedures were used with the predicate device: CAPILLARYS Hb A1c using the CAPILLARYS 2 FLEX-PIERCING instrument (K122101).

Both the new device MINICAP Hb A1c with the MINICAP FLEX-PIERCING and the predicate method, CAPILLARYS Hb A1c with the CAPILLARYS 2 FLEX-PIERCING instrument use the same technology of capillary electrophoresis of blood samples for Hb A1c analysis. The MINICAP HbA1c kit using the MINICAP FLEX-PIERCING instrument and the predicate device the CAPILLARYS Hb A1c kit used with the CAPILLARYS 2 FLEX-PIERCING instrument utilize EDTA collection tubes of (K2 and K3).

The SEBIA MINICAP Hb A1c procedure, performed with the MINICAP FLEX-PIERCING system was found to be substantially equivalent in function, use, safety, effectiveness and the performance to predicate devices described above.

The following tables A, B, C and D present the similarities and the differences.

Table A : SEBIA MINICAP Hb A1c kit used with the MINICAP FLEX PIERCING instrument as compared to the predicate CAP!LLARYS Hb A1c kit used with the CAPILLARYS 2 FLEX-PIERCING instrument

Table B: SEBIA Hb A1c CAPILLARY Calibrators used with the MINICAP Hb A1C kit and MINICAP FLEX-PIERCING Instrument to the predicate CAPILLARYS Hb A1c kit used with the CAPILLARYS 2 FLEX-PIERCING instrument.

Table C: SEBIA Hb A1c CAPILLARY Controls used with the MINICAP Hb A1c kit and MINICAP FLEX-PIERCING instrument as compared to the predicate devices CAPILLARYS Hb A1c kit used with the CAPILLARYS 2 FLEX-PIERCING instrument.

Table D: SEBIA MINICAP FLEX-PIERCING Instrument to the predicate the CAPILLARYS 2 FLEX-PIERCING instrument.

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### Table A

SEBIA MINICAP Hb A1c kit used with the MINICAP FLEX PIERCING instrument as compared to the predicate CAPILLARYS Hb A1c kit used with the CAPILLARYS 2 FLEX-PIERCING instrument.

| | SEBIA CAPILLARYS Hb A1c technique with<br>CAPILLARYS 2 FLEX-PIERCING instrument | SEBIA MINICAP Hb A1c technique with MINICAP<br>FLEX-PIERCING instrument |
|----------------------------------------------------------------|-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
| Intended Use | The CAPILLARYS Hb A1c kit is designed for<br>separation and quantification of the HbA1c glycated<br>fraction of hemoglobin in human blood, by capillary<br>electrophoresis in alkaline buffer (pH 9.4) with the<br>CAPILLARYS 2 FLEX-PIERCING instrument.<br>Measurement of hemoglobin A1c is effective in<br>monitoring long-term glycemic control in individuals<br>with diabetes mellitus. The CAPILLARYS Hb A1c kit<br>is designed for Professional Use Only.<br><br>For In Vitro Diagnostic Use. | The MINICAP Hb A1c kit is designed for separation<br>and quantification of the HbA1c glycated fraction of<br>hemoglobin in human blood, by capillary<br>electrophoresis in alkaline buffer (pH 9.4) with the<br>MINICAP FLEX-PIERCING instrument.<br>Measurement of hemoglobin A1c is effective in<br>monitoring long-term glycemic control in individuals<br>with diabetes mellitus. The MINICAP Hb A1c kit is<br>designed for Professional Use Only.<br><br>For In Vitro Diagnostic Use. |
| Separation<br>system | Free solution capillary electrophoresis (FSCE):<br>protein separation in an alkaline buffer (pH 9.4)<br>according to their charge, to the electrolyte pH and<br>electroosmotic flow.<br>Fast separation and good resolution.<br>Electrophoregrams show separated fractions<br>according to their charge. | Same |
| Instrument | SEBIA CAPILLARYS 2 FLEX-PIERCING instrument,<br>PN 1227 | SEBIA MINICAP FLEX-PIERCING instrument, PN<br>1232 |
| Picture | Image: SEBIA CAPILLARYS 2 FLEX-PIERCING instrument | Image: SEBIA MINICAP FLEX-PIERCING instrument |
| Interface | PC interface | Same |
| Absorbance wave<br>length | 415 nm | Same |
| Software | SEBIA PHORESIS™ software | Same |
| Number of<br>separation units | 8 parallel capillaries | 2 parallel capillaries |
| Calibration | Yes | Yes |
| Sample type | Whole blood in capped tube | Same |
| Samples<br>identification | Yes (Bar code reading on both sample racks and<br>tubes) | Yes (Bar code reading on sample tubes) |
| Hemolysis | Performed automatically by the instrument | Same |
| Introduction of<br>the samples into<br>the automatic<br>system | Continuous loading using sample racks | Continuous loading on the rotating sampler |
| Analysis<br>throughput | 40 analyses / hour | 7.6 analyses / hour |
| Collection tubes | Tubes with K2EDTA or K3EDTA anticoagulant | Same |
| | SEBIA CAPILLARYS Hb A1c technique with<br>CAPILLARYS 2 FLEX-PIERCING instrument | SEBIA MINICAP Hb A1c technique with MINICAP<br>FLEX-PIERCING instrument |
| Reagent | CAPILLARYS Hb A1c Kit (PN 2015) : | MINICAP Hb A1c Kit (PN 2215) : |
| | Buffer | Buffer |
| | Hemolyzing solution | Hemolyzin…