INSTRINSIC FACTOR AB, CALIBRATORS, AND QC ON THE ACCESS IMMUNOASSAY SYSTEMS, MODELS 387992, 387993, 387999

K033603 · Beckman Coulter, Inc. · LIG · Dec 19, 2003 · Clinical Chemistry

Device Facts

• Record ID: K033603
• Device Name: INSTRINSIC FACTOR AB, CALIBRATORS, AND QC ON THE ACCESS IMMUNOASSAY SYSTEMS, MODELS 387992, 387993, 387999
• Applicant: Beckman Coulter, Inc.
• Product Code: LIG · Clinical Chemistry
• Decision Date: Dec 19, 2003
• Decision: SESE
• Submission Type: Traditional
• Regulation: 21 CFR 862.1810
• Device Class: Class 2

Indications for Use

The Access Intrinsic Factor Ab assay is for the detection of intrinsic factor antibody in human serum and plasma using the Access Immunoassay systems. Intrinsic factor antibodies are measured as an aid in the diagnosis of pernicious anemia.

Device Story

Access Intrinsic Factor Ab assay is a paramagnetic particle, chemiluminescent immunoassay used on Access Immunoassay Systems (Access, Access 2, Synchron LXi 725, UniCel Dxl 800). Input: human serum or plasma. Principle: binding of intrinsic factor antibody to alkaline phosphatase enzyme-conjugated intrinsic factor, followed by competitive binding reaction with monoclonal antibody on paramagnetic solid-phase particles. Detection: dioxetane-based chemiluminescent substrate measuring light production. Used in clinical laboratory settings by trained personnel. Output: quantitative or qualitative detection of intrinsic factor antibodies. Results aid clinicians in diagnosing pernicious anemia. Benefits: automated, non-radioactive alternative to traditional radioimmunoassays.

Clinical Evidence

Clinical study used samples from healthy subjects to establish an upper reference limit (1.20 AU/mL) and ROC analysis to determine a positive cutoff (1.53 AU/mL). Method comparison study of 127 patient samples (including 67 positive) compared the Access assay to a predicate radioimmunoassay. Results: 100% negative agreement, 96.4% positive agreement, and 92.1% overall agreement. Analytical studies included imprecision (total CV 4.6-5.3%), dilution linearity, and specificity testing (no interference from bilirubin, hemoglobin, albumin, triglycerides, or vitamin B12 < 1500).

Technological Characteristics

Paramagnetic particle-based chemiluminescent immunoassay. Utilizes alkaline phosphatase enzyme conjugate and dioxetane-based substrate. Solid phase: paramagnetic particles. Detection: light measurement via chemiluminescence. System: Access Immunoassay Systems (Access, Access 2, Synchron LXi 725, UniCel Dxl 800). Reagents/calibrators stable for 56 days; calibration stable for 14 days.

Indications for Use

Indicated for the detection of intrinsic factor antibody in human serum and plasma as an aid in the diagnosis of pernicious anemia.

Regulatory Classification

Identification

A vitamin B12 test system is a device intended to measure vitamin B12 in serum, plasma, and urine. Measurements obtained by this device are used in the diagnosis and treatment of anemias of gastrointestinal malabsorption.

Predicate Devices

• DPC IFbAb (k811927)

Submission Summary (Full Text)

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510(k) SUBSTANTIAL EQUIVALENCE DETERMINATION
DECISION SUMMARY
DEVICE ONLY TEMPLATE

A. 510(k) Number:
k033603

B. Analyte:
Type I Intrinsic factor antibody

C. Type of Test:
Competitive immunoenzymatic assay

D. Applicant:
Beckman Coulter

E. Proprietary and Established Names:
Intrinsic Factor Ab Assay, Calibrators, and QC material

F. Regulatory Information:
1. Regulation section:
21 CFR § 862.1810, Vitamin B₁₂ test system
862.1660, Quality control material
862.1150, Calibrator
2. Classification:
Class II, I, II
3. Product Code:
LIG, Radioassay, Intrinsic factor blocking antibody
JJX, Single analyte controls
JIT, Secondary calibrator
4. Panel:
Clinical Chemistry (75)

G. Intended Use:
1. Indication(s) for use:
The Access Intrinsic Factor Ab assay is for the detection of intrinsic factor antibody in human serum and plasma using the Access Immunoassay systems.

Intrinsic factor antibodies are measured as an aid in the diagnosis of pernicious anemia.

2. Special condition for use statement(s):
For professional use only.

Plasma samples used in this assay should be heparinized plasma samples only.

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3. Special instrument Requirements:

Access® Immunoassay System (comprised of Access, Access 2, Synchron LXi 725, and UniCel Dxl 800 analyzers)

H. Device Description:

The Intrinsic Factor Ab Assay consists of 3 wet reagents provided in a ready-to-use test pack for use on Access Immunoassay analyzers. The reagents are as follows:

1. Paramagnetic particles coated with goat anti-mouse IgG, mouse monoclonal anti-intrinsic factor antibodies, buffer, surfactant, and preservative.
2. Porcine intrinsic factor-alkaline phosphatase conjugate complex, buffer, surfactant, and preservative.
3. Buffered protein blocking solution with preservative.

I. Substantial Equivalence Information:

1. Predicate device name(s): DPC IF bAb
2. Predicate K number(s): K811927
3. Comparison with predicate:

| Similarities | | |
| --- | --- | --- |
| Item | Device | Predicate |
| Intended Use | Detection of intrinsic factor antibody in human serum and plasma | Detection of intrinsic factor blocking antibody in serum |
| Antibody measured | Intrinsic Factor | Intrinsic Factor |
| Format | Competitive | Competitive |
| Differences | | |
| Item | Device | Predicate |
| Methodology | Automated enzyme immunoassay | Manual radioimmunoassay |
| Calibration | Single point stored calibration curve | Single point, no stored calibration |
| Sample Type | Serum and plasma | Serum |
| Antibody Type | Monoclonal | None |

J. Standard/Guidance Document Referenced (if applicable):

ANSI/ISO/ASQC A3534-1-1993

NCCLS EP7-P - Interference Testing in Clinical Chemistry

NCCLS EP5-A - Evaluation of Precision Performance of Clinical Chemistry Devices

NCCLS H18-A2 - Procedures for the Handling and Processing of Blood Specimens

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## K. Test Principle:

To measure Intrinsic factor antibody, sample is mixed with the blocking solution and the intrinsic factor-alkaline phosphatase conjugate solution. The mixture is incubated to promote binding of the intrinsic factor conjugate by intrinsic factor antibodies in the sample. The anti-intrinsic factor antibody-coated paramagnetic beads are then added and incubated. The antibodies on the beads compete with the antibodies in the sample for labeled intrinsic factor conjugate. The unbound enzyme conjugate is washed away as the beads are held in a magnetic field. Chemiluminescent enzyme substrate is added and the emitted light is measured. Light production is inversely proportional to the concentration of intrinsic factor antibody in the sample (reported as $\mathrm{AU / mL} =$ Antibody Units/mL).

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

### 1. Analytical performance:

#### a. Precision/Reproducibility:

To evaluate assay imprecision, five levels of in-house controls were assayed in duplicate for 28 days (according to NCCLS EP5-A guidelines). Results are summarized below.

| Sample | Mean (AU/mL) | Within Run SD | Within Run %CV | Between Run SD | Between Run %CV | Total %CV |
| --- | --- | --- | --- | --- | --- | --- |
| Level 1 | 1.06 | 0.02 | 1.46 | 0.05 | 4.36 | 4.59 |
| Level 2 | 1.27 | 0.02 | 1.28 | 0.06 | 4.58 | 4.75 |
| Level 3 | 1.61 | 0.02 | 1.34 | 0.08 | 4.69 | 4.88 |
| Level 4 | 4.63 | 0.07 | 1.48 | 0.23 | 5.07 | 5.28 |
| Level 5 | 14.19 | 0.21 | 1.46 | 0.67 | 4.71 | 4.93 |

Dilution recovery was evaluated using 2 serum samples. Dilutions were made and assayed in replicates of 4. The results (below) show that samples go from positive to negative upon dilution.

| Serum Sample 1 | | |
| --- | --- | --- |
| Dilution Factor | Concentration (AU/mL) | Results |
| Neat | 25.67 | Positive |
| 1/5 | 15.71 | Positive |
| 1/10 | 10.90 | Positive |
| 1/25 | 3.45 | Positive |
| 1/50 | 1.72 | Positive |
| 1/100 | 1.47 | Equivocal |
| 1/250 | 1.11 | Negative |
| 1/500 | 1.04 | Negative |
| 1/1000 | 1.02 | Negative |
| Serum Sample 2 | | |
| --- | --- | --- |
| Dilution Factor | Concentration (AU/mL) | Results |
| Neat | 35.29 | Positive |
| 1/5 | 20.66 | Positive |
| 1/10 | 13.24 | Positive |
| 1/25 | 3.76 | Positive |
| 1/50 | 2.14 | Positive |
| 1/100 | 1.6 | Positive |
| 1/250 | 1.2 | Equivocal |
| 1/500 | 1.08 | Negative |
| 1/1000 | 1.03 | Negative |

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b. Linearity/assay reportable range:

This assay reports a ratio that is assigned based on a zero calibrator, and is designated as Antibody Units/mL (AU/mL). The zero calibrator is assigned the value of 1.00 AU/mL. Values less than 0.90 AU/mL should be inspected for contamination.

c. Traceability (controls, calibrators, or method):

The commercially available calibrators are traceable to an internal reference calibrator that is prepared gravimetrically. The Access Intrinsic Factor Calibrator is a single level liquid calibrator containing buffer, human serum albumin, sodium azide, and ProClin300. The calibrator is assigned a value of 1.00 AU/mL in a series of qualification assays. The calibrator value is provided to the user on a card that can be bar-coded into the Access analyzer systems. Calibration is specified every 14 days.

The Access Intrinsic Factor Ab QC material consists of two levels of control material with targeted values of 1.05 and 2.00 AU/mL. The controls consist of intrinsic factor antibody in human serum with mouse proteins, sodium azide and ProClin 300.

Stability studies for the calibrator and control materials are described and are currently ongoing.

d. Detection limit:

Not applicable. This is not a quantitative test.

e. Analytical specificity:

Normal human serum was spiked with the compounds below and compared to the neat sample values to determine potential interference. No significant interference was seen at the levels tested.

| Potential Interferent | Concentration Added | Expected (AU/mL) | Observed (AU/mL) | Mean % Recovery |
| --- | --- | --- | --- | --- |
| Hemoglobin | 500 mg/dL | 0.95 | 1.02 | 107 % |
| Bilirubin | 20 mg/dL | 0.95 | 0.99 | 104 % |
| Lipemia (triolein) | 1800 mg/dL | 0.95 | 0.97 | 102 % |
| HSA | 9 % | 1.01 | 1.00 | 99 % |

Potential interference of naturally occurring vitamin $\mathrm{B}_{12}$ was evaluated by measuring Intrinsic factor antibody in 12 patient samples with vitamin $\mathrm{B}_{12}$ concentrations between 700 and $1600\mathrm{pg / mL}$ (as measured by the sponsor's vitamin $\mathrm{B}_{12}$ assay) in duplicate and comparing those values to Intrinsic factor antibody concentrations obtained with a commercially available RIA kit. No significant interference was seen up to $1500\mathrm{pg / mL}$ of vitamin $\mathrm{B}_{12}$ .

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Potential interference of free vitamin $\mathrm{B}_{12}$ levels was evaluated by spiking various levels of vitamin B12 (as measured by the sponsor's Vitamin $\mathrm{B}_{12}$ assay) into the assay's calibrator (contains no intrinsic factor antibody - $\mathrm{AU/mL} = 1.00$ ). Results are summarized below:

| Sample # | Vitamin B12 (pg/mL) | Access IFAb Result (AU/mL) | Access Result |
| --- | --- | --- | --- |
| 1 | 1.5 | 1.00 | Negative |
| 2 | 104 | 1.05 | Negative |
| 3 | 163 | 1.12 | Negative |
| 4 | 189 | 1.15 | Negative |
| 5 | 201 | 1.19 | Negative |
| 6 | 255 | 1.23 | Equivocal |
| 7 | 279 | 1.27 | Equivocal |
| 8 | 292 | 1.30 | Equivocal |
| 9 | 322 | 1.32 | Equivocal |
| 10 | 348 | 1.40 | Equivocal |
| 11 | 365 | 1.41 | Equivocal |
| 12 | 444 | 1.47 | Equivocal |
| 13 | 447 | 1.54 | Positive |
| 14 | 471 | 1.58 | Positive |
| 15 | 521 | 1.59 | Positive |
| 16 | 530 | 1.71 | Positive |

Negative samples did not test positive until free vitamin $\mathrm{B}_{12}$ levels were more than $444~\mathrm{pg / mL}$ . This information is in the product labeling along with a caution that this assay should not be used on patients that have received vitamin $\mathrm{B}_{12}$ injection therapy within the previous week.

To assess the effect of other interferences, 87 patient samples were assayed with the device and compared to measurements by the predicate device. The three discrepant samples (in the rheumatoid arthritis patients) were found to be positive in a different commercially available intrinsic factor RIA kit. A statement regarding possible interference with autoimmune disease was added to the labeling. Results are summarized below:

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| Disease | # of Samples | Access IFAb | | Commercial RIA | |
| --- | --- | --- | --- | --- | --- |
| | | Positive | Negative | Positive | Negative |
| Rheumatoid arthritis | 15 | 5 | 10 | 2 | 13 |
| Diabetes | 4 | 0 | 4 | 0 | 4 |
| Graves Disease | 5 | 0 | 5 | 0 | 5 |
| Hashimoto’s Thyroiditis | 5 | 0 | 5 | 0 | 5 |
| Thyroglobulin Ab | 5 | 0 | 5 | 0 | 5 |
| Low TSH | 4 | 0 | 4 | 0 | 4 |
| HAMA | 24 | 0 | 24 | 0 | 24 |
| Heterophiles | 25 | 0 | 25 | 0 | 25 |
| Totals | 87 | 5 | 82 | 2 | 85 |
| Overall Agreement = 96.6 % | | | | | |

f. Assay cut-off:
Not applicable.

2. Comparison studies:

a. Method comparison with predicate device:

Sixty-seven (67) positive and 60 negative stored samples (as originally determined by the predicate device) were assayed by the device for Intrinsic Factor Antibodies. These samples were also re-assayed with the predicate device. The results are as follows:

| Access | Negative | Predicate Device | | |
| --- | --- | --- | --- | --- |
| | | Negative | Indeterminate | Positive |
| Negative | 61 | 6 | 2 | |
| Equivocal | 0 | 3 | 0 | |
| Positive | 0 | 2 | 53 | |

Negative Agreement: 100 %
95% CI = 94.1 % - 100 %

Positive Agreement: 96.4 %
95% CI = 87.5 % - 99.6 %

Overall Agreement: 92.1 %
95% CI = 86.0 % - 96.2 %

b. Matrix comparison:

To evaluate equivalency of serum and heparinized plasma in the assay, 48 matched serum and heparin plasma samples from apparently healthy adults were tested for each sample type. In addition, 20 samples were spiked with intrinsic factor antibodies to yield a positive result. 100% agreement of positive and negative results was observed.

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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:

The sponsor has defined three patient status levels with respect to the detection of Intrinsic factor antibodies. Results &lt;1.20 AU/mL are reported as Negative, results ≥1.20 and less than 1.53 AU/mL are reported as Equivocal, and results ≥1.53 AU/mL are reported as Positive. The negative cutoff (1.20 AU/mL) was determined from the 99th percentile URL and the positive cutoff (1.53 AU/mL) was determined as the point at which sensitivity and specificity were maximized. This was determined by ROC analysis of 499 samples ranging from 0.93 to 12.25 AU/mL. Sensitivity and specificity are relative to the predicate device.

5. Expected values/Reference range:

The Upper Reference Limit for normals (URL) was determined by measuring the Intrinsic factor antibody in serum samples from 200 apparently healthy individuals. The URL non-parametric 99th percentile was determined to be 1.20 AU/mL.

M. Conclusion:

I recommend that the Access Intrinsic Factor Ab Assay, Calibrators, and QC material assay is substantially equivalent to the legally marketed predicate device.