VITROS CHEMISTRY PRODUCTS CAFFN REAGENT KIT AND TDM PERFORMANCE VERIFIERS I, II AND III

K042520 · Ortho-Clinical Diagnostics, Inc. · KLS · Nov 30, 2004 · Clinical Toxicology

Device Facts

• Record ID: K042520
• Device Name: VITROS CHEMISTRY PRODUCTS CAFFN REAGENT KIT AND TDM PERFORMANCE VERIFIERS I, II AND III
• Applicant: Ortho-Clinical Diagnostics, Inc.
• Product Code: KLS · Clinical Toxicology
• Decision Date: Nov 30, 2004
• Decision: SESE
• Submission Type: Traditional
• Regulation: 21 CFR 862.3880
• Device Class: Class 2
• Attributes: Pediatric

Indications for Use

VITROS Chemistry Products CAFFN Reagent Kit: For in vitro diagnostic use only. VITROS Chemistry Products CAFFN Reagent Kit is used on the VITROS 5,1 FS Chemistry System to quantitatively measure caffeine (CAFFN) concentration in human serum and plasma of subjects undergoing therapy with caffeine, especially for cases of neonatal apnea. VITROS Chemistry Products TDM Performance Verifier I, II and III: For in vitro diagnostic use only. VITROS TDM Performance Verifier is an assayed control used to monitor performance of ACET, CRBM, DGXN, PHBR, PHYT and CAFFN on VITROS Chemistry Systems.

Device Story

VITROS Chemistry Products CAFFN Reagent Kit; quantitative in vitro diagnostic assay for caffeine concentration in human serum/plasma. Operates on VITROS 5,1 FS Chemistry System. Two-step competitive immunoassay; caffeine labeled with glucose-6-phosphate dehydrogenase (G6P-DH) competes with sample caffeine for sheep polyclonal antibody binding sites. G6P-DH converts NAD+ to NADH; absorbance measured spectrophotometrically at 340 nm. Absorbance change inversely proportional to caffeine concentration. Used in clinical laboratory settings by trained personnel. Output provides quantitative caffeine levels to monitor therapeutic drug concentrations; aids clinical decision-making for caffeine therapy management, specifically neonatal apnea. Includes assayed controls (VITROS TDM Performance Verifiers) to monitor system performance.

Clinical Evidence

No clinical trials performed. Evidence based on bench testing and method comparison. Method comparison study (n=122; 110 neonatal, 20 adult samples) against predicate yielded Y=0.9995X - 0.04 µg/mL, R=0.989. Precision studies (n=88) showed within-lab %CV of 4.4-6.5%. Linearity confirmed 0.7-32 µg/mL (R²=0.9931). Analytical sensitivity (LOD) 0.14-0.15 µg/mL. Interference testing per NCCLS EP7-A showed no significant bias for common endogenous substances or therapeutic drugs.

Technological Characteristics

Two-step competitive immunoassay. Reagents: sheep polyclonal antibodies, G6P-DH labeled caffeine, NAD+. Instrumentation: VITROS 5,1 FS Chemistry System. Measurement: Spectrophotometric (340 nm). Calibrators: 6 levels, traceable to USP Caffeine reference standard. Stability: 12 weeks (open/reconstituted at 2-8°C). Controls: Gravimetrically prepared from bovine serum. Standards: NCCLS EP9-A, EP5-A, EP7-A, EP6-A.

Indications for Use

Indicated for quantitative measurement of caffeine in human serum and plasma for patients undergoing caffeine therapy, particularly for neonatal apnea. TDM Performance Verifiers are indicated for monitoring assay performance of ACET, CRBM, DGXN, PHBR, PHYT, and CAFFN on VITROS Chemistry Systems.

Regulatory Classification

Identification

A theophylline test system is a device intended to measure theophylline (a drug used for stimulation of the muscles in the cardiovascular, respiratory, and central nervous systems) in serum and plasma. Measurements obtained by this device are used in the diagnosis and treatment of theophylline overdose or in monitoring levels of theophylline to ensure appropriate therapy.

Predicate Devices

• SYVA Emit Caffeine Assay (k853872)
• VITROS Chemistry Products TDM Performance Verifiers (k982649)

Submission Summary (Full Text)

{0}

1

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

A. 510(k) Number:
k042520

B. Purpose for Submission:
New Device

C. Measurand:
Caffeine

D. Type of Test:
Quantitative

E. Applicant:
Ortho-Clinical Diagnostics, Inc.

F. Proprietary and Established Names:
VITROS Chemistry Products CAFFN Reagent Kit
VITROS Chemistry Products TDM Performance Verifier I, II, and III

G. Regulatory Information:
1. Regulation section:
21 CFR 862.3800 Theophylline Test System
21 CFR 862.3280 Drug Mixture Control Material
2. Classification:
21 CFR 862.3800 Theophylline Test System, Class II.
21 CFR 862.3280 Controls, Class I, reserved.

{1}

3. Product code:
KLS
DIF

4. Panel:
91 (Toxicology), and 91 (Toxicology)

H. Intended Use:

1. Intended use(s):
See Indications for Use below.

2. Indication(s) for use:
For in vitro diagnostic use only. VITROS Chemistry Products CAFFN Reagent Kit is used on the VITROS 5,1 FS Chemistry System to quantitatively measure caffeine (CAFFN) concentration in human serum and plasma of subjects undergoing therapy with caffeine, especially for cases of neonatal apnea.
For in vitro diagnostic use only. VITROS TDM Performance Verifier is an assayed control used to monitor performance of ACET, CRBM, DGXN, PHYT, and CAFFN on VITROS Chemistry Systems.

3. Special conditions for use statement(s):
For Prescription Use Only.

4. Special instrument requirements:
VITROS 5,1 FS Chemistry System (previously cleared K031924)

I. Device Description:
The VITROS Chemistry Products CAFFN Reagent Kit is a three component dual chamber device.

Component one consists of lyophilized reagent 1 and reagent 2, six levels of calibrators and buffer concentrate. Reagent 1 in the VITROS CAFFN Reagent Kit is sheep polyclonal antibodies reactive to caffeine, glucose-6-phosphate (Na-G6P), Nicotinamide Adenine Dinucleotide (NAD) and rabbit serum albumin. Reagent 2 in the VITROS CAFFN Reagent Kit is caffeine labeled with glucose-6-phosphate dehydrogenase (G6P-DH), glucose-6-phosphate (Na-G6P) and rabbit serum albumin.

2

{2}

Component two is the VITROS CAFFN Verifiers I, II and III, which are a set of 3 verifiers (6 vials- 2 mL each) and are required for the VITROS CAFFN Reagent Kit.

Component three is the Diluent Pack 3 is a dual chamber that is used to dilute samples and uses processed water and a specialty diluent prepared from processed human serum.

The VITROS CAFFN Kit and the Diluent Pack 3 was tested using FDA approved methods and was found nonreactive for hepatitis B surface antigen (HBsAg), antibody to HCV and antibody to HIV. This information is indicated in the labeling.

## J. Substantial Equivalence Information:

1. Predicate device name(s):

SYVA Emit Caffeine Assay

VITROS Chemistry Products TDM Performance Verifiers, I, II, and III

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

SYVA Emit Caffeine Assay k853872

VITROS Chemistry Products TDM Performance Verifiers – k982649

3. Comparison with predicate:

VITROS Chemistry Products CAFFN Reagent Kit

| Similarities | | |
| --- | --- | --- |
| Item | Device | Predicate |
| Range | 1- 30 μg/mL | 1- 30 μg/mL |
| Calibrator | 6 levels included | 6 levels included |
| Stability | Opened: 12 weeks | Opened: 12 weeks |
| Storage | 2-8°C | 2-8°C |

{3}

4

| Differences | | |
| --- | --- | --- |
| Item | Device | Predicate |
| Intended Use | For in vitro diagnostic use only. VITROS Chemistry Products CAFFN Reagent Kit is used on the VITROS 5,1 FS Chemistry System to quantitatively measure caffeine (CAFFN) concentration in human serum and plasma of subjects undergoing therapy with caffeine, especially for cases of neonatal apnea. | Quantitative measurement of caffeine as a metabolite. |
| Sample | Serum and Plasma | Serum |
| Instrumentation | VITROS 5,1 FS Chemistry System | SYVA-30R Biochemical System |

VITROS Chemistry Products TDM Performance Verifiers

| Similarities | | |
| --- | --- | --- |
| Item | Device | Predicate |
| Intended Use | For in vitro diagnostic use only. VITROS TDM Performance Verifier is an assayed control used to monitor performance of ACET, CRBM, DGXN, PHYT, and CAFFN on VITROS Chemistry Systems. | For in vitro diagnostic use only. VITROS TDM Performance Verifier is an assayed control use to monitor performance on VITROS Chemistry System. |
| Matrix | Serum | Serum |
| Levels | I, II and III | I, II and III |
| Differences | | |
| --- | --- | --- |
| Item | Device | Predicate |
| Instrumentation | VITROS 5,1 FS Chemistry System | SYVA-30R Biochemical System |
| Constituents | Addition of Caffeine(CAFFN), Digoxin (DGXN) and Phenytoin(PHYT) to the already cleared Acetaminophen (ACET), Carbamazepine(CRBM) and Phenobarbital (PHBR). | Acetaminophen (ACET), Carbamazepine (CRBM), Phenobarbital (PHBR). |

{4}

5

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

NCCLS document EP9-A. Method Comparison and Bias Estimation Using Patient Samples; Approved Guideline- Second Edition.

NCCLS document EP5-A. Method Comparison of Precision Performance of Clinical Chemistry Devices; Approved Guideline.

NCCLS document EP7-A. Interference Testing in Clinical Chemistry; Approved Guideline.

NCCLS document EP6-A. Evaluation of the Linearity of Quantitative Measurement Procedure: A Statistical Approach; Approved Guideline.

L. Test Principle:

The VITROS Chemistry Products CAFFN Reagent Kit is a two-step reaction that quantitatively measures caffeine. Caffeine labeled with a glucose-6-phosphate dehydrogenase (G6P-DH) competes with caffeine in a sample for antibody binding sites. G6P-DH conversion of Nicotinamide Adenine Dinucleotide (NAD+) to NADH and the sample is read spectrophotometrically at 340 nm. The absorbance change is proportional to the caffeine concentration in the sample.

M. Performance Characteristics (if/when applicable):

1. Analytical performance:

a. Precision/Reproducibility:

Two lots of the VITROS Chemistry Products CAFFN Reagent were tested in duplicate on two separate VITROS 5,1 FS Systems. Same day runs were separated by a two hour minimum. Each run assayed three control sera in duplicate for mean concentrations of 2.62, 12.55 and 21.30 µg/mL. Mean values for the samples that were tested for within-day, within-lab precision, within-lab %CV and the number of observations are shown in the table below.

| Mean Concentration | Within Day SD | Within Lab SD | Within Lab %CV | # of Observation |
| --- | --- | --- | --- | --- |
| 2.62 µg/mL | 0.092 | 0.171 | 6.5 % | 88 |
| 12.55 µg/mL | 0.291 | 0.553 | 4.4 % | 88 |
| 21.30 µg/mL | 0.536 | 0.957 | 4.5 % | 84 |

Water from the VITROS Chemistry Products FS Diluent Pack 3 (Specialty Diluent/Water) was tested for dilution ability and accuracy with two VITROS

{5}

Chemistry Products Reagent lots. Ten aliquots of normal human serum samples spiked with caffeine stock solutions ranging from 22.5 to 30 µg/mL. The samples were diluted 1:2 and 1:4 with water from the Diluent Pack 3. The samples were tested in triplicate and a mean was calculated for the neat samples and the diluted samples. For Lot 1, the percent recovery for the 1:2 and 1:4 diluted samples was 91.5% and 86.5% respectively. For Lot 2, the recovery for the 1:2 and 1:4 diluted samples was 94.5% and 89.8% respectively.

b. Linearity/assay reportable range:

Two caffeine spiked pools of serum high pool (55 µg/mL) and a low pool (near zero µg/mL) were mixed to create 15 additional pools of intermediate concentrations. Five determinations for each fluid level and three determinations of VITROS Chemistry Products TDM Performance Verifiers were conducted with two lots of CAFFN Reagents on two VITROS 5,1 FS Chemistry Systems. A plot of the observed results (between 0.7-32 µg/mL) versus the expected results yielded an equation of Y=0.4345X-0.2018 and an R² of 0.9931 between 0.7-32 µg/mL. Acceptable linearity was defined as when the bias between predicted and calculated caffeine concentrations were within predetermined acceptance limits.

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

The VITROS Chemistry Products CAFFN Reagent Kit Calibrators are traceable to a USP Caffeine reference standard (catalogue # 1085003). Primary calibrators are prepared by the manufacturer and the six levels are confirmed by GC/MS or HPLC methods. The sponsor confirms the calibrator values using another test system. One hundred clinical serum samples are assigned values from this run. The serum samples are then used to calibrate the VITROS test system, and to assign values to the sponsor's master lot of calibrators. This procedure ensures that matrix effects associated with both methods are taken into account in the value transfer process.

The VITROS Chemistry Products were found to have a stability of 12 weeks when open and/or reconstituted at 2-8 °C. On board stability of the VITROS Chemistry Products CAFFN Reagent Kit is 12 weeks if the system is turned on and 20 minutes if the system is turned off. This claim is similar to the predicate's.

The VITROS TDM Performance Caffeine (CAFFN), Digoxin (DGXN) and Phenytoin (PHYT) Verifiers are included in this submission. The VITROS TDM Performance Verifiers are prepared gravimetrically from bovine serum, therapeutic drugs, inorganic salts and preservatives. The VITROS TDM Performance Verifiers have an open vial stability of 7 days when stored at 2-8 °C. The VITROS TDM Performance Verifiers Control values are

6

{6}

assigned using two instruments and include two calibrations, multiple samples of each reagent lot and 32 replicates of the performance verifier. An allowable range is then applied around these reagent lot-specific target values to establish upper and lower control limits. The allowable range reflects estimates of expected within-lab and lab-to-lab variability of properly operating VITROS 5,1 FS Chemistry Systems. A range of means was determined on 5 VITROS 5,1 FS Chemistry System in different laboratories for 10 days, 2 runs per day and 2 replicates per run for each performance verifier. A pooled SD was determined using the total SD's from each site to give a +/- range as 3 times the pooled SD.

## d. Detection limit:

The lower limit of detection was studied using 4 determinations with 2 reagent lots and 2 calibrator lots on two VITROS 5,1 FS Chemistry Systems. Ten serum samples from normal adult human donors that contained little or no caffeine were analyzed in triplicates using a low level calibrator, a serum base pool and saline. It was determined that the low level calibrator had no caffeine. Serum caffeine concentrations from 3 of the 10 donors were greater than 0.5 µg/mL and were excluded from the study. The lower limit of detection was calculated using the following equation:

$$
\mathrm{LD} = 3.3 \times \left(\frac{\text{Calibration error Variance (SD)}^2 + \text{Pooled Replicate Variance (SD)}^2}{1/2}\right).
$$

The lower limit of detection ranged from 0.14 µg/mL to 0.15 µg/mL and supports that claim of 0.2 µg/mL.

## e. Analytical specificity:

The substances listed in the table below at the concentrations shown, were tested according to NCCLS Protocol EP7-A with VITROS CAFFN Reagent and a serum pool at a caffeine concentration of 7 µg/mL (with the exception of Bilirubin, Hemoglobin and Intralipid). An off the clot human serum pool spiked with caffeine to create caffeine concentrations of 0.6 µg/mL was spiked with a caffeine stock of 10,000 µg/mL to create 2 base pools of caffeine concentrations of approximately 5 and 20 µg/mL. These two pools were used in testing bilirubin, hemoglobin and Intralipid. The results are shown in the table below:

| Compound | Concentration | |
| --- | --- | --- |
| Bilirubin | 60 mg/dL | 1026 µmol/L |
| 8-Chlorotheophylline | 100 µg/mL | 466 µmol/L |
| 1,3-Dimethyluric Acid | 100 µg/mL | 510 µol/L |
| Dyphylline | 100 µg/mL | 393 µmol/L |
| Hemoglobin | 1000 mg/dL | 10 g/L |

{7}

| Compound | Concentration | |
| --- | --- | --- |
| Hypoxanthine | 100 μg/mL | 735 μmol/L |
| Intralipid | 1000 mg/dL | 10 g/L |
| 3-Isobutyl 1- | 50 μg/mL | 225 μmol/L |
| 1-Methyluric Acid | 100 μg/mL | 549 μmol/L |
| 3-Methyluric Acid | 100 μg/mL | 549 μmol/L |
| 1-Methylxanthine | 100 μg/mL | 602 μmol/L |
| 3-Methylxanthine | 100 μg/mL | 602 μmol/L |
| 7-Methylxanthine | 100 μg/mL | 602 μmol/L |
| Paraxanthine | 4 μg/mL | 22 μmol/L |
| Phenobarbital | 100 μg/mL | 431 μmol/L |
| Theobromine | 30 μg/mL | 167 μmol/L |
| Theophylline | 50 μg/mL | 278 μmol/L |
| 1,3,7-Trimethyluric Acid | 30 μg/mL | 143 μmol/L |
| Urea | 200 μg/mL | 1.2 μmol/L |
| Xanthine | 100 μg/mL | 657 μmol/L |

Interference was determined by calculating bias using the following equation:

Bias= Mean conc. of test substance pool – Mean conc. of Control pool.

The samples tested for Bilirubin, Hemoglobin and Intralipid interference with a serum pool at a caffeine concentration of approximately 5 and 20 μg/mL and was found not to interfere (bias <3.91 μg/mL). All other substances listed in the table above were tested with VITROS CAFFN Reagent Kit and a serum pool with caffeine concentration of 7 μg/mL and was found not to interfere (bias of <1.92 μg/mL).

f. Assay cut-off:

N/A

2. Comparison studies:

a. Method comparison with predicate device:

The sponsor states that studies were based on NCCLS Guideline EP9-A2 Method Comparison and Bias Estimation Using Patient Samples.

A total of 122 samples, sixty-six human serum and fifty-six plasma samples (110 neonatal and 20 adult samples), were assayed in triplicate with two lots of both the VITROS CAFFN assay and the Syva EMIT Caffeine assay. Caffeine concentrations for both the neonatal and adult samples ranged from

{8}

1.09 to 23.45 µg/mL. A plot of the correlation between the VITROS CAFFN Assay and the Syva EMIT Caffeine assay and yielded a least squares linear regression of Y= 0.9995X -0.04 µg/mL and a correlation coefficient of 0.989.

b. Matrix comparison:

To determine which specimen types are suitable for analysis with the assay, the sponsor conducted a study involving various specimens from different Becton Dickinson tubes.

Ten whole blood samples were collected in red top serum tubes and seperated. The serum was spiked with caffeine and transferred to various tube types. The samples were studied to determine the appropriate specimen types and proper fill levels for the VITROS CAFFN Assay. The specimens were tested in triplicate with two lots of the VITROS CAFFN Reagent kit. The following table shows specimen types and fill levels for the specimens examined.

| Sample Type | Fill Levels Collected |
| --- | --- |
| Serum (Red Top) | Full |
| Serum Separator (SST) | Full, ½ Full |
| Li-Heparin Plasma | ¼ Full |
| Li-Heparin Plasma Separator (PST) | Full |
| Sodium Citrate | Full |
| EDTA Plasma | ¼ Full |
| Sodium Fluoride Potassium Oxalate | Full |

Serum and plasma samples were evaluated by paired-difference testing. The data was tabulated and the bias between the mean value (n=3) for each test condition was compared ot the mean of the serum value. The bias was calculated as :

Bias = Test Condition Prediction – Serum Sample Value

Serum (red top) was used as the reference becasue it is the specimen matrix used to establish overall accuracy of the method.

The acceptabne criteria are based on the caffeine concentration in the serum sample :

| Caffeine Concentration (μg/mL) | Bias Limits |
| --- | --- |
| Less than 5 μg/mL | +/- 1.160 μg/mL |
| Greater than 5 μg/mL | +/- 0.1661[caffeine] + 0.2540 |

Serum samples (red top tubes), samples collected in full serum separator tubes (SST), and tubes containing the anticoagulant lithium heparin were within the sponsorss predetermined acceptance criteria for the VITROS Chemistry

{9}

Products CAFFN assay.

The sponsor reported that serum samples collected in partially filled serum separator (1/2 filled) tubes may show a positive bias ranging from 3 to 11%. Serum samples collected in tubes containing EDTA did not meet acceptance criteria. Although serum samples collected in tubes containing Sodium Citrate or Sodium Flouride Potassium Oxalate bias values were within were within predetermined acceptance criteria, the sponsor makes no claim for these specimen types.

3. Clinical studies:

a. Clinical Sensitivity:

N/A

b. Clinical specificity:

N/A

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

N/A

4. Clinical cut-off:

N/A

5. Expected values/Reference range:

Reference ranges are presented below.

| Classification | Conventional Unit (μg/mL) | SI Units (μmol/L) | Alternate Units (mg/L) |
| --- | --- | --- | --- |
| Therapeutic | 8 -20 | 41 - 103 | 8 - 20 |
| Toxic (Critical) | >50 | >258 | >50 |

These reference intervals are in published literature recommended by the National Academy of Clinical Biochemistry (NACB). Use of the same ranges is further supported by the close agreement observed in the linear regression plot from the method comparison study involving the predicate assay.

N. Proposed Labeling:

The labeling is sufficient and it satisfies the requirements of 21 CFR Part 809.10.

{10}

O. Conclusion:

The submitted information in this premarket notification is complete and supports a substantial equivalence decision.

11