ACE HEMOGLOBIN ALC(HBALC) REAGENT, ACE CEDIA T UPTAKE REAGENT, ACE T4 REAGENT, ACE FERRITIN REAGENT

K113437 · Alfa Wassermann Diagnostic Technologies, Inc. · LCP · Aug 6, 2012 · Hematology

Device Facts

• Record ID: K113437
• Device Name: ACE HEMOGLOBIN ALC(HBALC) REAGENT, ACE CEDIA T UPTAKE REAGENT, ACE T4 REAGENT, ACE FERRITIN REAGENT
• Applicant: Alfa Wassermann Diagnostic Technologies, Inc.
• Product Code: LCP · Hematology
• Decision Date: Aug 6, 2012
• Decision: SESE
• Submission Type: Traditional
• Regulation: 21 CFR 864.7470
• Device Class: Class 2

Indications for Use

The ACE Axcel Clinical Chemistry System is an automated, discrete, bench-top, random access analyzer that is intended for in vitro diagnostic use in the quantitative determination of constituents in blood and other fluids. ACE Hemoglobin A1c (HbA1c) Reagent is intended for the quantitative determination of hemoglobin A1c (µmol/L) and total hemoglobin (g/dL) in human EDTA whole blood for the calculation of percent hemoglobin A1c using the ACE Axcel Clinical Chemistry System. The test is intended for use in clinical laboratories or physician office laboratories to monitor long term blood glucose control in individuals with diabetes mellitus. For in vitro diagnostic use only. The ACE CEDIA T Uptake homogenous enzyme immunoassay is intended for the quantitative determination of unoccupied binding sites of thyroxine-binding proteins in serum using the ACE Axcel Clinical Chemistry System. Measurements of triiodothyronine uptake are used in the diagnosis and treatment of thyroid disorders. This test is intended for use in clinical laboratories or physician office laboratories. For in vitro diagnostic use only. The ACE T4 Reagent is intended for the quantitative determination of total thyroxine (T4) concentration in serum using the ACE Axcel Clinical Chemistry System. Total thyroxine measurements are used in the diagnosis and treatment of thyroid diseases. This test is intended for use in clinical laboratories or physician office laboratories. For in vitro diagnostic use only. The ACE Ferritin Reagent is intended for the quantitative determination of ferritin concentration in serum using the ACE Axcel Clinical Chemistry System. Measurements of ferritin aid in the diagnosis of diseases affecting iron metabolism, such as hemochromatosis (iron overload) and iron deficiency anemia. This test is intended for use in clinical laboratories or physician office laboratories. For in vitro diagnostic use only.

Device Story

System uses ACE Axcel Clinical Chemistry System to perform quantitative immunoassays on human EDTA whole blood (HbA1c) or serum (T Uptake, T4, Ferritin). HbA1c assay uses latex agglutination inhibition; total hemoglobin measured via alkaline hematin conversion. T Uptake and T4 use homogeneous enzyme immunoassays (CEDIA or G6PD-labeled competition). Ferritin uses latex agglutination turbidimetry. System automates sample pretreatment, reagent mixing, and spectrophotometric absorbance measurement at specific wavelengths (e.g., 592 nm, 340 nm). Results are calculated via calibration curves and reported to clinicians for monitoring diabetes, thyroid function, and iron status. Used in clinical labs and physician office labs by trained personnel. Output aids clinical decision-making regarding disease diagnosis and treatment monitoring.

Clinical Evidence

No clinical data provided; substantial equivalence supported by bench testing and performance validation of the analytical assays on the ACE Axcel system.

Technological Characteristics

Quantitative immunoassays; latex agglutination (HbA1c, Ferritin) and homogeneous enzyme immunoassay (T Uptake, T4). Reagents include mouse monoclonal antibodies, latex particles, and enzymes (β-galactosidase, G6PD). Analysis performed at 37°C on ACE Axcel Clinical Chemistry System. Calibration via multi-point or linear regression curves. Sterilization not applicable (reagents).

Indications for Use

Indicated for quantitative determination of HbA1c/total hemoglobin (diabetes monitoring), T-uptake (thyroid disorders), total T4 (thyroid diseases), and ferritin (iron metabolism disorders) in human blood/serum. Intended for use in clinical or physician office laboratories.

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

• Alfa Wassermann ACE Clinical Chemistry System (K931786)
• ACE Reagents (K063306, K981375, K981377, K050944)

Submission Summary (Full Text)

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510(k) SUBSTANTIAL EQUIVALENCE DETERMINATION
DECISION SUMMARY
ASSAY TEMPLATE

A. 510(k) Number:
k113437

B. Purpose for Submission:
New Device

C. Measurand:
Glycosylated Hemoglobin
Triiodothyronine Uptake
Total Thyroxine
Ferritin

D. Type of Test:
Quantitative, immunoassay

E. Applicant:
Alfa Wassermann Diagnostic Technologies, LLC

F. Proprietary and Established Names:
ACE Hemoglobin A1c (HbA1c) Reagent
ACE CEDIA T Uptake Reagent
ACE T4 Reagent
ACE Ferritin Reagent

G. Regulatory Information:
1. Regulation section:
21 C.F.R. § 864.7470
21 C.F.R. § 862.1715

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21 C.F.R. § 862.1700

21 C.F.R. § 866.5340

2. Classification:

Class II, class II meets limitations to exemptions 862.9(c)(9), class II, class II respectively

3. Product code:

LCP, KHQ, KLI and DBF respectively

4. Panel:

Chemistry 75

H. Intended Use:

1. Intended use(s):

See Indications for Use below

2. Indication(s) for use:

ACE Hemoglobin A1c (HbA1c) Reagent is intended for the quantitative determination of hemoglobin A1c (μmol/L) and total hemoglobin (g/dL) in human EDTA whole blood for the calculation of percent hemoglobin A1c using the ACE Axcel Clinical Chemistry System. The test is intended for use in clinical laboratories or physician office laboratories to monitor long term blood glucose control in individuals with diabetes mellitus. For in vitro diagnostic use only.

The ACE CEDIA T Uptake homogenous enzyme immunoassay is intended for the quantitative determination of unoccupied binding sites of thyroxine-binding proteins in serum using the ACE Axcel Clinical Chemistry System. Measurements of triiodothyronine uptake are used in the diagnosis and treatment of thyroid disorders. This test is intended for use in clinical laboratories or physician office laboratories. For in vitro diagnostic use only.

The ACE T4 Reagent is intended for the quantitative determination of total thyroxine (T4) concentration in serum using the ACE Axcel Clinical Chemistry System. Total thyroxine measurements are used in the diagnosis and treatment of thyroid diseases. This test is intended for use in clinical laboratories or physician office laboratories. For in vitro diagnostic use only.

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The ACE Ferritin Reagent is intended for the quantitative determination of ferritin concentration in serum using the ACE Axcel Clinical Chemistry System. Measurements of ferritin aid in the diagnosis of diseases affecting iron metabolism, such as hemochromatosis (iron overload) and iron deficiency anemia. This test is intended for use in clinical laboratories or physician office laboratories. For in vitro diagnostic use only.

3. Special conditions for use statement(s):

For prescription use and point-of-care use

4. Special instrument requirements:

Alfa Wassermann ACE Axcel Clinical Chemistry System

I. Device Description:

The ACE HbA1c Reagent is composed of four reagent bottles (Hemoglobin Denaturant, Total Hemoglobin Reagent, HbA1c Agglutinator Reagent and HbA1c Antibody Reagent). The Hemoglobin Denaturant contains: pepsin and buffer (pH 2.4). The Total Hemoglobin Reagent contains: sodium hydroxide (pH 13) and Triton (Octylphenoxypolyethoxyethanol). The HbA1c agglutinator reagent contains: HbA1c hapten covalently attached to a polymer, bovine serum albumin and buffer (pH 2.0). The HbA1c Antibody Reagent contains: HbA1c antibody (mouse) coupled particles, bovine serum albumin and buffer.

The ACE CEDIA T Uptake Reagent is composed of two dot-coded, empty reagent bottles for the Enzyme Acceptor (EA) Reagent and the Enzyme Donor (ED) Reagent. The EA Reagent contains enzyme acceptor (microbial), 0.111 g/L; phosphate buffer; buffer salts; stabilizers; preservative; detergent. The ED Reagent contains enzyme donor (microbial)-thyroxine conjugate, 0.44 mg/L; o-nitro-β-D-galactopyranoside, 3.27 g/L; phosphate buffer; buffer salts; stabilizers; detergent; preservative.

The ACE T4 Reagent is composed of two reagent bottles (Antibody/Substrate Reagent and Enzyme Conjugate Reagent). The Antibody/Substrate Reagent (R1) contains: mouse monoclonal anti-thyroxine antibody, 8-anilino-1-naphthalene sulfonic acid, glucose-6-phosphate, nicotinamide adenine dinucleotide and Tris buffer. The Enzyme Conjugate Reagent (R2) contains: glucose-6-phosphate dehydrogenase labeled with thyroxine and Tris buffer.

The ACE Ferritin Reagent is composed of two dot-coded, empty reagent bottles for the

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Ferritin Buffer and the Ferritin Antibody Reagent. The Ferritin Buffer (R1) contains a glycine buffer. The Ferritin Antibody Reagent (R2) contains a suspension of latex particles sensitized with rabbit anti-human ferritin antibody.

All products derived from blood are prepared exclusively from the blood of donors tested individually by FDA-approved methods and found to be negative for HbsAg and antibodies to HCV and HIV1/HIV2.

## J. Substantial Equivalence Information:

1. Predicate device name(s):

ACE Clinical Chemistry System, ACE HbA1c Reagent

ACE Clinical Chemistry System, ACE CEDIA T UP Reagent

ACE Clinical Chemistry System, ACE T4 Reagent

ACE Clinical Chemistry System, ACE Ferritin Reagent

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

k063306, k981375, k981377 and k050944

3. Comparison with predicate:

| | New Device | Predicate Device |
| --- | --- | --- |
| 510(k) # | k113437 | k931786 (ACE) |
| Company | Alfa Wassermann Diagnostic Technologies, LLC | Alfa Wassermann Diagnostic Technologies, LLC |
| Name | ACE Axcel Clinical Chemistry System, ACE Hemoglobin A1c Reagent | ACE Clinical Chemistry System, ACE Hemoglobin A1c Reagent |
| Intended Use/Indication for Use | Same | Intended for the quantitative determination of hemoglobin A1c (μmol/L) and total hemoglobin (g/dL) in human EDTA whole blood for the calculation of percent hemoglobin A1c. This test is intended for use in clinical laboratories or physician office laboratories to monitor long term blood glucose control in individuals with diabetes mellitus. For in vitro diagnostic use only. |
| Instrument Platforms | ACE Axcel Clinical Chemistry System | ACE Clinical Chemistry System |
| Calibration | Same | 6 point for HbA1c, 1 point for total hemoglobin |
| Calibration Stability | Same | 5 Days |
| Use of Controls | Same | Two levels of control per day |
| Basic Principle | Same | Antibody-coated latex agglutination inhibition |
| Measurement Type | Same | Quantitative ratio of HbA1c to total hemoglobin, expressed as percent |

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| | New Device | Predicate Device |
| --- | --- | --- |
| 510(k) # | k113437 | k931786 (ACE) |
| Company | Alfa Wassermann Diagnostic Technologies, LLC | Alfa Wassermann Diagnostic Technologies, LLC |
| Name | ACE Axcel Clinical Chemistry System, ACE Hemoglobin A1c Reagent | ACE Clinical Chemistry System, ACE Hemoglobin A1c Reagent |
| Reactive Ingredients | Same | Porcine pepsin
Sodium hydroxide
HbA1c hapten polymer
Bovine serum albumin
HbA1c antibody (mouse) coupled particles
Buffer |
| Sample Type | Same | Whole blood (EDTA) |
| | New Device | Predicate Device |
| --- | --- | --- |
| 510(k) # | K113437 | K931786 (ACE) |
| Company | Alfa Wassermann Diagnostic Technologies, LLC | Alfa Wassermann Diagnostic Technologies, LLC |
| Name | ACE Axcel Clinical Chemistry System, ACE CEDIA T Uptake Reagent | ACE Clinical Chemistry System, ACE CEDIA T Uptake Reagent |
| Intended Use/Indications for Use | Same | Intended for the quantitative determination of unoccupied binding sites of thyroxine-binding proteins in human serum. For in vitro diagnostic use only. |
| Instrument Platforms | ACE Axcel Clinical Chemistry System | ACE® Clinical Chemistry System |
| Calibration | Same. | The system utilizes data reduction from least-squares linear regression on a 2 point calibration. |
| Calibration Stability | Same | 24 hours without correction and 4 days when updated daily with distilled or deionized water as the corrector. |
| On-Board Stability | Same | 30 Days |
| Use of Controls | Same | Two levels of controls per day |
| Basic Principle | Same | CEDIA® Cloned Enzyme Donor Immunoassay |
| Measurement Type | Same | Homogenous enzyme immunoassay |
| Reactive Ingredients | Same | Enzyme acceptor(microbial)
Phosphate buffer
Buffer salts
Enzyme donor(microbial)-thyroxine conjugate
o-Nitrophenyl-β-D-galactopyranoside |

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| | New Device | Predicate Device |
| --- | --- | --- |
| 510(k) # | K113437 | K931786 (ACE) |
| Company | Alfa Wassermann Diagnostic Technologies, LLC | Alfa Wassermann Diagnostic Technologies, LLC |
| Name | ACE Axcel Clinical Chemistry System, ACE CEDIA T Uptake Reagent | ACE Clinical Chemistry System, ACE CEDIA T Uptake Reagent |
| Dimensions | Same | Bottles with total volumes of 49 mL and 11 mL |
| Analysis Temperature | Same | 37°C |
| Sample Type | Serum | Serum |
| | New Device | Predicate Device |
| --- | --- | --- |
| 510(k) # | K113437 | K931786 (ACE) |
| Company | Alfa Wassermann Diagnostic Technologies, LLC | Alfa Wassermann Diagnostic Technologies, LLC |
| Name | ACE Axcel Clinical Chemistry System, ACE T4 Reagent | ACE Clinical Chemistry System, ACE T4 Reagent |
| Intended Use/Indications for Use | Same | Intended for the quantitative determination total thyroxine (T4) in serum. For in vitro diagnostic use only. |
| Instrument Platforms | ACE Axcel Clinical Chemistry System | ACE and ACE Alera® Clinical Chemistry Systems |
| Calibration | Same | The system utilizes data reduction calculated from a non-linear curve fit on a multi-point calibration |
| Calibration Stability | Same | 5 Days |
| On-Board Stability | Same | 30 Days |
| Use of Controls | Same | Two levels of controls per day |
| Basic Principle | Same | Homogenous enzyme immunoassay |
| Measurement Type | Same | Delta |
| Reactive Ingredients | Same | Mouse monoclonal anti-thyroxine antibody 8-Anilino-1-naphthalene sulfonic acid Glucose-6-phosphate Nicotinamide adenine dinucleotide Tris buffer |
| Analysis Temperature | Same | 37°C |
| Reaction Type | Same | Delta |
| Sample Type | Same | Serum |
| | New Device | Predicate Device |
| --- | --- | --- |
| 510(k) # | K113437 | K931786 (ACE) |

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| Company | Alfa Wassermann Diagnostic Technologies, LLC | Alfa Wassermann Diagnostic Technologies, LLC |
| --- | --- | --- |
| Name | ACE Axcel Clinical Chemistry System, ACE Ferritin Reagent | ACE Clinical Chemistry System, ACE Ferritin Reagent |
| Intended Use/Indications for Use | Same | Intended for the quantitative determination of ferritin (an iron-storing protein) in serum. The measurement of ferritin is useful in assessing diseases affecting iron metabolism, such as hemochromatosis (iron overload) and iron deficiency anemia. For in vitro diagnostic use only. |
| Instrument Platforms | ACE Axcel Clinical Chemistry System | ACE and ACE *Alera*^{®} Clinical Chemistry Systems |
| Calibration | Same | The system utilizes data reduction calculated from a non-linear curve fit using a multi-point calibration. The use of Ferritin Calibrators is recommended. |
| Calibration Stability | Same | 7 Days |
| On-Board Stability | Same | 30 Days |
| Use of Controls | Same | Two levels of control per day |
| Basic Principle | Same | Latex agglutination |
| Measurement Type | Same | Serum Ferritin, in the presence of anti-ferritin conjugated latex microparticles and a buffer promoting aggregation, initiates an antigen-antibody reaction, resulting in the agglutination of the latex microparticles. The magnitude of the absorbance change is proportional to the Ferritin concentration in the sample. The concentration is determined by calculation from a calibration curve generated using calibrators containing known Ferritin concentrations. |
| Reactive Ingredients | Same | Latex coupled to rabbit anti-human ferritin antibody Glycine buffer |
| Analysis Temperature | Same | 37°C |
| Reaction Type | Same | Delta |
| Sample Type | Same | Serum |

K. Standard/Guidance Document Referenced (if applicable):
Method Comparison and Bias Estimation Using Patient Samples; Approved Guideline – Second Edition (CLSI EP9-A2)
Evaluation of the Linearity of Quantitative Measuring Procedures: A Statistical Approach; Approved Guideline (CLSI EP6-A)

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Evaluation of Precision Performance of Quantitative Measurement Methods; Approved Guideline – Second Edition (CLSI EP5-A2)

Determination of Limits of Detection and Limits of Quantification; Approved Guideline (CLSI EP17-A)

L. Test Principle:

Prior to the ACE Hemoglobin A1c (HbA1c) Reagent assay, whole blood samples require a pretreatment step, which is done on-board the analyzer. The red blood cells in the sample are lysed by the Hemoglobin Denaturant and the hemoglobin chains are hydrolyzed. For determination of HbA1c, a latex agglutination inhibition assay is used. In the absence of HbA1c in the sample, the agglutinator (synthetic polymer containing the immunoreactive portion of HbA1c) in the HbA1c Agglutinator Reagent and the antibody-coated microparticles in the HbA1c Antibody Reagent will agglutinate. The presence of HbA1c in the sample competes for the antibody binding sites and inhibits agglutination. The increase in absorbance, monitored monochromatically at 592 nm, is inversely proportional to the HbA1c present in the sample. For the determination of total hemoglobin, all hemoglobin derivatives in the sample are converted to alkaline hematin. The reaction produces a green colored solution, which is measured bichromatically at 573 nm/692 nm. The intensity of color produced is directly proportional to the total hemoglobin concentration in the sample. The concentrations of both HbA1c and total hemoglobin are measured, the ratio is calculated and the result reported as percent HbA1c.

The CEDIA T Uptake assay uses recombinant DNA technology to produce a unique homogeneous enzyme immunoassay system. The assay is based the bacterial enzyme β-galactosidase, which has been genetically engineered into two inactive fragments. These fragments spontaneously re-associate to form fully active enzyme which, in the assay format, cleaves a substrate, generating a color change that can be measured spectrophotometrically. In the assay, enzyme donor thyroxine conjugate binds directly to the unoccupied thyroxine-binding sites in the sample, preventing the spontaneous re-association of the enzyme fragments to form the active enzyme. Thus, thyroxine-binding proteins regulate the amount of β-galactosidase formed from the reassembly of the remaining donor and enzyme acceptor as monitored by the hydrolysis of the substrate o-nitrophenyl-β-galactopyranoside.

The ACE T4 Assay is a homogeneous enzyme immunoassay using ready-to-use liquid ACE T4 Reagent. The assay uses 8-anilino-1-naphthalene sulfonic acid (ANS) to dissociate thyroxine from the plasma binding proteins. Using specific antibodies to thyroxine, this assay is based on the competition of glucose-6-phosphate dehydrogenase (G6PD) labeled thyroxine and the dissociated thyroxine in the sample for a fixed amount of specific antibody binding sites. In the absence of thyroxine from the sample, the thyroxine labeled G6PD in the second reagent is bound by the specific antibody in the first reagent, inhibiting the enzyme's activity. The enzyme G6PD catalyzes the oxidation of glucose-6-phosphate (G6P) with nicotinamide adenine dinucleotide (NAD⁺) to form 6-phosphogluconate and reduced nicotinamide adenine dinucleotide (NADH). NADH strongly absorbs at 340 nm whereas NAD⁺ does not. The rate of conversion, determined by measuring the increase in absorbance bichromatically at 340

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nm/505 nm during a fixed time interval, is directly proportional to the amount of thyroxine in the sample. The concentration of thyroxine is determined automatically by the ACE Clinical Chemistry System using a logarithmic calibration curve established with calibrators, which are provided separately.

In the Ferritin Assay, serum ferritin, in the presence of anti-ferritin conjugated latex microparticles, and a buffer promoting aggregation, initiates an antigen-antibody reaction, resulting in the agglutination of the latex microparticles. The agglutination is detected turbidimetrically by an absorbance change measured at a wavelength of 592 nm. The magnitude of the absorbance change is proportional to the ferritin concentration in the sample.

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

### 1. Analytical performance:

#### a. Precision/Reproducibility:

Studies were carried out in accordance with CLSI Guidance Document EP5-A2. Precision was determined on the ACE Axcel Clinical Chemistry System at three levels of EDTA whole blood sample containing HbA1c. The three levels were assayed at least 2 times per run, 2 runs per day, for a total of 22 days. The precision was found to be:

| Sample 1
Mean 5.31% HbA1c | Within
Run | Between
Run | Between
Day | Total |
| --- | --- | --- | --- | --- |
| Standard Deviation,
%HbA1c | 0.08 | 0.06 | 0.08 | 0.13 |
| Coefficient of Variation | 1.5% | 1.2% | 1.6% | 2.5% |
| Sample 2
Mean 8.28% HbA1c | Within
Run | Between
Run | Between
Day | Total |
| --- | --- | --- | --- | --- |
| Standard Deviation,
%HbA1c | 0.16 | 0.00 | 0.14 | 0.21 |
| Coefficient of Variation | 2.0% | 0.0% | 1.6% | 2.5% |

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Sample 3
Mean 11.98% HbA1c

| Standard Deviation, %HbA1c | Within Run | Between Run | Between Day | Total |
| --- | --- | --- | --- | --- |
| | 0.28 | 0.00 | 0.22 | 0.36 |
| Coefficient of Variation | 2.3% | 0.0% | 1.9% | 3.0% |

Further precision studies were done at Alfa Wassermann and in three separate Physician Office Laboratory (POL) sites. The studies consisted of running three EDTA whole blood samples with varying levels of HbA1c in triplicate on five different days.

| | | | %CV or SD (%HbA1c) | |
| --- | --- | --- | --- | --- |
| Lab | Sample | Mean | Within-Run | Total |
| POL 1 | 1 | 5.01 | 0.13 SD | 0.13 SD |
| | | | 2.6% | 2.6% |
| POL 2 | 1 | 5.11 | 0.10 SD | 0.18 SD |
| | | | 2.0% | 3.6% |
| POL 3 | 1 | 5.01 | 0.11 SD | 0.11 SD |
| | | | 2.2% | 2.2% |
| | | | | |
| POL 1 | 2 | 7.65 | 0.08 SD | 0.14 SD |
| | | | 1.0% | 1.8% |
| POL 2 | 2 | 7.93 | 0.12 SD | 0.23 SD |
| | | | 1.5% | 2.9% |
| POL 3 | 2 | 7.64 | 0.15 SD | 0.18 SD |
| | | | 2.0% | 2.4% |
| | | | | |
| POL 1 | 3 | 10.23 | 0.20 SD | 0.26 SD |
| | | | 2.0% | 2.5% |
| POL 2 | 3 | 10.67 | 0.19 SD | 0.34 SD |
| | | | 1.8% | 3.1% |
| POL 3 | 3 | 10.25 | 0.12 SD | 0.18 SD |
| | | | 1.2% | 1.8% |

## T Uptake

Studies were carried out in accordance with CLSI Guidance Document EP5-A2. Precision was determined on the ACE Axcel Clinical Chemistry System at four patient serum samples T Uptake levels. The four levels were assayed at least 2 times per run, 2 runs per day, for a total of 21 days. The precision was found to be:

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| **Sample 1**
Mean 39.74% T Uptake | Within Run | Between Run | Between Day | Total |
| --- | --- | --- | --- | --- |
| Standard Deviation, % | 0.92 | 0.53 | 1.35 | 1.71 |
| Coefficient of Variation | 2.3% | 1.3% | 3.4% | 4.3% |
| **Sample 2**
Mean 32.88% T Uptake | Within Run | Between Run | Between Day | Total |
| --- | --- | --- | --- | --- |
| Standard Deviation, % | 0.65 | 0.54 | 1.00 | 1.31 |
| Coefficient of Variation | 2.0% | 1.6% | 3.0% | 4.0% |
| **Sample 3***
Mean 26.82% T Uptake | Within Run | Between Run | Between Day | Total |
| --- | --- | --- | --- | --- |
| Standard Deviation, % | 0.85 | 0.37 | 1.07 | 1.42 |
| Coefficient of Variation | 3.2% | 1.4% | 4.0% | 5.3% |

*Data collected for 22 days

| **Sample 4***
Mean 30.67% T Uptake | Within Run | Between Run | Between Day | Total |
| --- | --- | --- | --- | --- |
| Standard Deviation, % | 0.81 | 0.00 | 1.20 | 1.44 |
| Coefficient of Variation | 2.6% | 0.0% | 3.9% | 4.7% |

Further precision studies were done at Alfa Wassermann and in three separate Physician Office Laboratory (POL) sites. The studies consisted of running three patient serum samples with varying levels of T Uptake in triplicate on five different days.

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| | | | %CV or SD (%T Uptake) | |
| --- | --- | --- | --- | --- |
| Lab | Sample | Mean | Within-Run | Total |
| POL 1 | 1 | 38.30 | 0.78 SD | 2.19 SD |
| | | | 2.0% | 5.7% |
| POL 2 | 1 | 41.49 | 0.98 SD | 1.71 SD |
| | | | 2.4% | 4.1% |
| POL 3 | 1 | 39.04 | 0.58 SD | 0.89 SD |
| | | | 1.5% | 2.3% |
| | | | | |
| POL 1 | 2 | 32.12 | 0.78 SD | 1.22 SD |
| | | | 2.4% | 3.8% |
| POL 2 | 2 | 34.05 | 0.57 SD | 1.04 SD |
| | | | 1.7% | 3.1% |
| POL 3 | 2 | 32.81 | 0.91 SD | 1.09 SD |
| | | | 2.8% | 3.3% |
| | | | | |
| POL 1 | 3 | 28.86 | 0.99 SD | 1.40 SD |
| | | | 3.4% | 4.9% |
| POL 2 | 3 | 30.64 | 1.50 SD | 1.50 SD |
| | | | 4.9% | 4.9% |
| POL 3 | 3 | 29.81 | 0.57 SD | 0.74 SD |
| | | | 1.9% | 2.5% |

## Thyroxine

Studies were carried out in accordance with CLSI Guidance Document EP5-A2. Precision was determined on the ACE Axcel Clinical Chemistry System at four serum thyroxine levels. The four levels were assayed at least 2 times per run, 2 runs per day, for a total of 21 days. The precision was found to be:

| Sample 1
Mean 10.28 μg/dL
Thyroxine | Within Run | Between Run | Between Day | Total |
| --- | --- | --- | --- | --- |
| Standard Deviation, μg/dL | 0.24 | 0.00 | 0.20 | 0.32 |
| Coefficient of Variation | 2.3% | 0.0% | 2.0% | 3.1% |

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| **Sample 2**
Mean 16.40 μg/dL
Thyroxine | Within
Run | Between
Run | Between
Day | Total |
| --- | --- | --- | --- | --- |
| Standard Deviation, μg/dL | 0.42 | 0.07 | 0.17 | 0.45 |
| Coefficient of Variation | 2.5% | 0.4% | 1.0% | 2.8% |
| **Sample 3***
Mean 18.15 μg/dL
Thyroxine | Within
Run | Between
Run | Between
Day | Total |
| --- | --- | --- | --- | --- |
| Standard Deviation, μg/dL | 0.87 | 0.00 | 0.11 | 0.88 |
| Coefficient of Variation | 4.8% | 0.0% | 0.6% | 4.8% |

*Data collected for 22 days

| **Sample 4***
Mean 6.98 μg/dL
Thyroxine | Within
Run | Between
Run | Between
Day | Total |
| --- | --- | --- | --- | --- |
| Standard Deviation, μg/dL | 0.18 | 0.00 | 0.17 | 0.25 |
| Coefficient of Variation | 2.6% | 0.0% | 2.5% | 3.6% |

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Further precision studies were done at Alfa Wassermann and in three separate Physician Office Laboratory (POL) sites. The studies consisted of running three patient serum samples with varying levels of thyroxine in triplicate on five different days.

| | | | %CV or SD (μg/dL) | |
| --- | --- | --- | --- | --- |
| Lab | Sample | Mean | Within-Run | Total |
| POL 1 | 1 | 10.21 | 0.24 SD | 0.27 SD |
| | | | 2.4% | 2.6% |
| POL 2 | 1 | 10.49 | 0.21 SD | 0.23 SD |
| | | | 2.0% | 2.2% |
| POL 3 | 1 | 10.29 | 0.15 SD | 0.23 SD |
| | | | 1.5% | 2.2% |
| | | | | |
| POL 1 | 2 | 16.81 | 0.45 SD | 0.52 SD |
| | | | 2.7% | 3.1% |
| POL 2 | 2 | 16.52 | 0.81 SD | 1.01 SD |
| | | | 4.9% | 6.1% |
| POL 3 | 2 | 16.83 | 0.26 SD | 0.35 SD |
| | | | 1.5% | 2.1% |
| | | | | |
| POL 1 | 3 | 17.94 | 0.39 SD | 0.48 SD |
| | | | 2.2% | 2.7% |
| POL 2 | 3 | 17.90 | 0.48 SD | 0.74 SD |
| | | | 2.7% | 4.1% |
| POL 3 | 3 | 18.40 | 0.36 SD | 0.38 SD |
| | | | 2.0% | 2.1% |

Ferritin

Studies were carried out in accordance with CLSI Guidance Document EP5-A2. Precision was determined on the ACE Axcel Clinical chemistry system at four serum ferritin levels. The four levels were assayed at least 2 times per run, 2 runs per day, for a total of 21 days. The precision was found to be:

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15

| **Sample 1**
Mean 49.6 ng/dL Ferritin | Within
Run | Between
Run | Between
Day | Total |
| --- | --- | --- | --- | --- |
| Standard Deviation, ng/dL | 2.1 | 0.0 | 2.0 | 2.9 |
| Coefficient of Variation | 4.3% | 0.0% | 4.1% | 5.9% |
| **Sample 2**
Mean 381.8 ng/dL Ferritin | Within
Run | Between
Run | Between
Day | Total |
| --- | --- | --- | --- | --- |
| Standard Deviation, ng/dL | 4.5 | 2.0 | 7.5 | 9.0 |
| Coefficient of Variation | 1.2% | 0.5% | 2.0% | 2.4% |
| **Sample 3***
Mean 547.4 ng/dL Ferritin | Within
Run | Between
Run | Between
Day | Total |
| --- | --- | --- | --- | --- |
| Standard Deviation, ng/dL | 23.8 | 0.0 | 28.7 | 37.3 |
| Coefficient of Variation | 4.3% | 0.0% | 5.2% | 6.8% |

*Data collected for 22 days

| **Sample 4***
Mean 47.0 ng/dL Ferritin | Within
Run | Between
Run | Between
Day | Total |
| --- | --- | --- | --- | --- |
| Standard Deviation, ng/dL | 2.0 | 0.0 | 1.8 | 2.7 |
| Coefficient of Variation | 4.3% | 0.0% | 3.9% | 5.8% |

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Further precision studies were done at Alfa Wassermann and in three separate Physician Office Laboratory (POL) sites. The studies consisted of running three patient serum samples with varying levels of ferritin in triplicate on five different days.

| | | | %CV or SD (unit) | |
| --- | --- | --- | --- | --- |
| Lab | Sample | Mean | Within-Run | Total |
| POL 1 | 1 | 52.3 | 2.2 SD | 2.3 SD |
| | | | 4.2% | 4.4% |
| POL 2 | 1 | 52.0 | 2.0 SD | 2.0 SD |
| | | | 3.8% | 3.8% |
| POL 3 | 1 | 51.2 | 2.2 SD | 2.2 SD |
| | | | 4.2% | 4.2% |
| | | | | |
| POL 1 | 2 | 387.6 | 4.7 SD | 5.6 SD |
| | | | 1.2% | 1.4% |
| POL 2 | 2 | 381.8 | 4.7 SD | 5.0 SD |
| | | | 1.2% | 1.3% |
| POL 3 | 2 | 381.1 | 4.2 SD | 7.1 SD |
| | | | 1.1% | 1.9% |
| | | | | |
| POL 1 | 3 | 519.3 | 18.2 SD | 18.2 SD |
| | | | 3.5% | 3.5% |
| POL 2 | 3 | 651.9 | 30.7 SD | 45.2 SD |
| | | | 4.7% | 6.9% |
| POL 3 | 3 | 646.4 | 22.5 SD | 29.5 SD |
| | | | 3.5% | 4.6% |

# b. Linearity/assay reportable range:

# HbA1c :

For HbA1c, commercial linearity materials with low, medium and high levels of HbA1c, were mixed in different known proportions. The mid and high samples, along with the 9 mixtures, were run using the on-board pretreatment method, in an identical manner to patient samples (total samples, $n = 11$ ). The 11 samples were then run in triplicate on the ACE Axcel Clinical Chemistry System using the ACE Hemoglobin A1c Reagent. The assigned values of the low and high samples were assigned by running on the ACE, prior to the

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performing the linearity study.

Linear regression analysis of the assigned value (x-axis) versus measured value (y-axis) demonstrates that the HbA1c assay is linear from 4.23 to 14.2% HbA1c (y = 0.984x + 0.21, r² = 0.9967).

The sponsor claims a range of 4.2 – 13%

## T Uptake:

Using mixtures of the two calibrators, the sponsor evaluated the linearity of 11 evenly spaces samples between 15%-50% were analyzed in triplicate on the ACE Axcel Clinical Chemistry System. T uptake was found to be linear between 16.4%-50%

Linear regression analysis was Y = 1.011x – 4.49

The sponsor claims a range of 16.4%-50%

## T4 Thyroxine

8 serum samples spiked to provide the range of thyroxine concentrations from 1.3 to 19.6 ug/dL µg/dL were analyzed in triplicate on the ACE Axcel Clinical Chemistry System. Linear regressions analysis was done and the results presented below:

Linear regression for the T4 assay is y = 1.005x + 0.8, R² = 0.9915.

The sponsor claims a range of 1.3 and 19.2 µg/dL

## Ferritin :

10 serum samples, spiked to provide the range of ferritin concentrations from 11.3 to 996.0 ng/dL were analyzed in triplicate on the ACE Axcel Clinical Chemistry System. Linear regressions analysis was done and the results presented below:

Linear regression for the Ferritin assay is y = 1.013 – 3.11, R² = 0.9967

The sponsor claims a range of 11 to 860 ng/dL

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

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Traceability:

For HbA1c, traceability is based on NGSP. Therefore, the following statement has been added to the Calibration section of the package insert (consistent with the predicate device package insert):

"The Hemoglobin A1c Assay has met the requirements of the National Glycohemoglobin Standardization Program. Certification from the National Glycohemoglobin Standardization Program (NGSP) requires annual renewal. Please refer to the NGSP website at http://www.ngsp.org/ to view the current list of NGSP certified methods."

For T4, traceability is based on the manufacturer's internal standards utilizing a T4 purity of at least than 98%. The manufacturer verifies each level of the Standard calibrators against the previous standard at ±2% with a replicate of n=10 per level. Additionally, the standard concentrations are verified with a minimum of two commercial methods.

T4 calibration curves generated from standards are verified utilizing commercial controls (n=3 per level for low, medium, and high concentrations). In addition, a correlation requirement between standards (current and new set) of equal or greater than 0.995 is established by the manufacturer utilizing patient samples (n=21) and CAP (College of American Pathologists) samples (n=10). A slope (0.95 – 1.05) and intercept (-0.70 – 0.70) are required.

For Cedia T Uptake, traceability is based on the manufacturer's in-house standards and method comparison testing. Calibrators and reagents are traceable to a required % purity of the original raw material. The Hypothyroid Calibrator level, 15%, is gravimetrically prepared from a Thyroxine Binding Globulin Stock and T-Uptake Serum, then lyophilized and stored at 2 -8 degree C. The Hyperthyroid Calibrator level, 50%, is gravimetrically prepared from a T4 Stock and T-Uptake Serum, then lyophilized and stored at 2 -8 degree C.

T Uptake calibration curves generated from kit calibrators are verified utilizing commercial controls (n=3 per level for low, medium, and high concentrations). In addition, a correlation requirement between standards (reference and on-test set) of equal or greater than 0.980 is established by the manufacturer utilizing patient samples (n=21). The sponsor's acceptance criteria are slope (0.93 – 1.07) and intercept (-2.00 – 2.00).

For the Ferritin Assay, the Ferritin calibrator is traceable to the WHO 1st International Ferritin Standard, 1984, lot 80/602".

18

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# Stability:

The ACE Reagents demonstrated on-board and calibration stability for the time periods in the following table, when stored on the ACE Axcel Clinical Chemistry System. The study protocols and acceptance criteria has been reviewed and found to be adequate. The sponsor proposed on-board and calibration stability claims on the ACE Axcel Clinical Chemistry System:

| Reagent | ACE Axcel On-Board | ACE Axcel Calibration |
| --- | --- | --- |
| | Stability (X Days) | Stability (X Days) |
| HbA1c | 30 | 5 |
| T Uptake | 30 | 4* |
| T4 | 30 | 5 |
| Ferritin | 30 | 7 |

*With daily correction

Real time stability studies are ongoing and performed according to CLSI EP25-A, Evaluation of Stability of In Vitro Diagnostic Method Products; Approved Guideline.

# d. Detection limit:

The sponsor performed the detection limit studies for ACE reagents on the ACE Axcel Clinical Chemistry System. Limit of detection for HbA1C is based on linearity studies and is $4.2\%$ . Limit of detection for T Uptake is based on linearity studies and is $16.4\%$ . The LoB, LoD and LoQ were determined according to CLSI EP 17-A for the Axcel Chemistry System for Ferritin and T4. For LoD, low samples and true blanks $(n = 60, 20$ reps per day) were tested over three days on two instruments. For LoQ, samples $(N = 40, 6$ reps per day) were tested in 5 separate runs over 5 days. The reportable range lower limits are as follows:

| Reagent | Units | ACE Axcel Reportable Range Lower Limits | ACE Axcel LoB | ACE Axcel LoD | ACE Axcel LoQ |
| --- | --- | --- | --- | --- | --- |
| T4 | μg/dL | 1.3 | 0.77 | 1.1 | 1.3 |
| Ferritin | ng/dL | 11.0 | 7.6 | 11.0 | 11.0 |

# e. Analytical specificity:

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Interference studies were performed to determine the effects from potential interferents. The ACE reagents were tested for interference caused by ascorbic acid, bilirubin, hemoglobin (with the exception of HbA1c) and lipemia/triglycerides and other potential interferents that were spiked into serum pools containing HbA1c, T4, T uptake and Ferritin reagents at normal and abnormal concentrations. All samples were tested in triplicate. Seven levels were tested for each interferent.

For all four reagents, studies to determine the effects of interferences were performed in accordance with CLSI Guidance Document EP7-A2.

Interference for each of these assays is considered to be significant by the sponsor if the analyte recovery changes by more than ±10%.

The level at which there is no significant interference for each analyte is listed below

For HbA1c:

| Interferent | No Significant Interference At or Below: |
| --- | --- |
| Unconjugated Bilirubin | 56 mg/dL |
| Triglycerides | 2000 mg/dL |
| Ascorbic Acid | 6 mg/dL |
| Acetaldehyde | 100 mg/dL |
| Acetylsalicylic Acid | 200 mg/dL |
| Sodium Cyanate (Carbamylated Hb) | 100 mg/dL |
| Sodium Fluoride | 1200 mg/dL |

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For T4:

| Interferent | No Significant Interference At or Below: |
| --- | --- |
| Unconjugated Bilirubin | 55 mg/dL |
| Hemolysis | 1000 mg/dL |
| Lipemia/Intralipid | 1000 mg/dL |
| Ascorbic Acid | 6 mg/dL |

For T Uptake:

| Interferent | No Significant Interference At or Below: |
| --- | --- |
| Unconjugated Bilirubin | 55 mg/dL |
| Hemolysis | 1000 mg/dL |
| Triglycerides | 1317 mg/dL |
| Ascorbic Acid | 6 mg/dL |

For Ferritin:

| Interferent | No Significant Interference At or Below: |
| --- | --- |
| Unconjugated Bilirubin | 62 mg/dL |
| Hemolysis | 1000 mg/dL |
| Triglycerides | 1310 mg/dL |
| Ascorbic Acid | 6 mg/dL |

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f. Assay cut-off:

Not Applicable

# 2. Comparison studies:

a. Method comparison with predicate device:

HbA1c

A series of 108 EDTA whole blood specimens (1 diluted) with HbA1c values ranging from 4.3 to $12.8\%$ were assayed in singlicate on the ACE Axcel Clinical Chemistry System using ACE HbA1c Reagent (y) and the ACE Clinical Chemistry System as the reference method (x). Least-squares regression analysis (Deming) yielded the following results:

| Regression Equation | y = 1.012x + 0.10 |
| --- | --- |
| Correlation Coefficient | 0.9912 |
| Std. Error Est. | 0.24 |
| Confidence Interval Slope | 0.986 to 1.038 |
| Confidence Interval Intercept | -0.07 to 0.27 |

# CEDIA T Uptake

A series of 113 serum specimens (2 diluted,6 spiked) with T Uptake values ranging from $16.4\%$ to $47.5\%$ were assayed in singlicate on the ACE Axcel Clinical Chemistry System using ACE CEDIA T Uptake Reagent (y) and the ACE Clinical Chemistry System as the reference method (x). Least-squares regression analysis (Deming) yielded the following results:

| Regression Equation | y = 1.032x - 0.29 |
| --- | --- |
| Correlation Coefficient | 0.9473 |
| Std. Error Est. | 1.65 |
| Confidence Interval Slope | 0.969 to 1.095 |
| Confidence Interval Intercept | -2.28 to 1.69 |

# T4

A series of 123 serum specimens (3 diluted,4 spiked) with T4 values ranging from 1.4 to $19.2\mu \mathrm{g / dL}$ were assayed in singlicate on the ACE Axcel Clinical Chemistry System using ACE T4 Reagent (y) and the ACE Clinical Chemistry System as the reference method (x). Least-squares regression analysis (Deming) yielded the following results:

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# Ferritin (Ferritin)

A series of 103 serum specimens( 2 diluted,2 spiked) with ferritin values ranging from 17 to $851\mathrm{ng / dL}$ were assayed in singlicate on the ACE Axcel Clinical Chemistry System using ACE Ferritin Reagent (y) and the ACE Clinical Chemistry System as the reference method (x). Least-squares regression analysis (Deming) yielded the following results:

| Regression Equation | y = 1.015x - 1.1 |
| --- | --- |
| Correlation Coefficient | 0.9988 |
| Std. Error Est. | 8.2 |
| Confidence Interval Slope | 1.005 to 1.024 |
| Confidence Interval Intercept | -3.5 to 1.2 |

Additional method comparison studies were performed at three Physician Office Laboratories, with four operators. Operators assayed serum samples ranging from $4.23 - 12.4\%$ HbA1c, $16.4\% -50.0\%$ T Uptake, $2.0 - 19.2~\mu \mathrm{g / dL}$ T4 and $12 - 792~\mathrm{ng / dL}$ ferritin on the Ace Axcel clinical chemistry analyzer and the ACE clinical chemistry System. The results are presented in the tables below:

| Reagent | POL | n | y = mx + b | r |
| --- | --- | --- | --- | --- |
| Hemoglobin A1c | 1 | 49 | y = 0.950x + 0.37 | 0.9920 |
| Hemoglobin A1c | 2 | 45 | y = 1.005x + 0.14 | 0.9793 |
| Hemoglobin A1c | 3 | 47 | y = 0.969x - 0.19 | 0.9929 |
| T Uptake | 1 | 49 | y = 0.961x + 1.04 | 0.9752 |
| T Uptake | 2 | 41 | y = 0.974x + 1.26 | 0.9625 |
| T Uptake | 3 | 48 | y = 0.953x + 0.41 | 0.9885 |
| T4 | 1 | 47 | y = 0.999x - 0.03 | 0.9929 |
| T4 | 2 | 41 | y = 1.022x - 0.05 | 0.9927 |
| T4 | 3 | 46 | y = 0.965x + 0.29 | 0.9910 |
| Ferritin | 1 | 53 | y = 1.046x - 8.2 | 0.9985 |
| Ferritin | 2 | 41 | y = 0.977x - 8.5 | 0.9926 |

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| Reagent | POL | n | y = mx + b | r |
| --- | --- | --- | --- | --- |
| Ferritin | 3 | 43 | y = 0.973x + 1.3 | 0.9996 |

b. Matrix comparison:

Not Applicable

3. Clinical studies:

a. Clinical Sensitivity:

Not Applicable

b. Clinical specificity:

Not Applicable

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

Not applicable

4. Clinical cut-off:

Not applicable

5. Expected values/Reference range:

| Reagent | Expected Values Range | Expected Values Support |
| --- | --- | --- |
| Ferritin ng/dL | 7-253 (M) 2-110 (F) | Kamiya Biomedical Company, K-ASSAY Ferritin, Package Insert, Rev. 2008-12-16 |
| T4 μg/dL | 4.8 – 13.0 | Tietz, et al., Clinical Guide to Laboratory Tests, Second Edition, 1990 |

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| Reagent | Expected Values Range | Expected Values Support |
| --- | --- | --- |
| T Uptake% | 24.3 – 39.0 | Roche Diagnostics GmbH, Cedia T Uptake Package Insert, Rev. 6, 2008-07 |
| HbA1c | Less than 5.7% | American Diabetes Association (2011). Diagnosis and Classification of Diabetes mellitus. Diabetes Care, 34(Suppl 1): S62–S69. |

# 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