VITROS Immunodiagnostic Products Total ß-hCG II Reagent Pack

K233581 · Ortho Clinical Diagnostics · DHA · May 8, 2024 · Clinical Chemistry

Device Facts

• Record ID: K233581
• Device Name: VITROS Immunodiagnostic Products Total ß-hCG II Reagent Pack
• Applicant: Ortho Clinical Diagnostics
• Product Code: DHA · Clinical Chemistry
• Decision Date: May 8, 2024
• Decision: SESE
• Submission Type: Traditional
• Regulation: 21 CFR 862.1155
• Device Class: Class 2

Indications for Use

For in vitro diagnostic use only. For the quantitative measurement of human chorionic gonadotropin (hCG) and its β-subunit in human serum and plasma (heparin and EDTA) using the VITROS 5600 Integrated System to aid in the early detection of pregnancy.

Device Story

The VITROS Immunodiagnostic Products Total β-hCG II Reagent Pack is an in vitro diagnostic reagent used on the VITROS 5600 Integrated System. It utilizes an immunometric sandwich immunoassay to measure human chorionic gonadotropin (hCG) and its β-subunit in human serum and plasma. The device inputs patient samples into microwells coated with biotinylated mouse monoclonal anti-β-hCG antibody; these bind to hCG in the sample. A Horseradish Peroxidase (HRP)-labeled mouse monoclonal anti-β-hCG conjugate is added to form a sandwich. After washing, a luminogenic substrate and electron transfer agent are added; HRP catalyzes a light-producing oxidation reaction. The system measures the light signal, which is directly proportional to the hCG concentration. The output is a quantitative hCG value used by clinicians to aid in the early detection of pregnancy. The device is intended for use in clinical laboratory settings.

Clinical Evidence

Bench testing only. Precision was evaluated per CLSI EP05-A3 (20-day and instrument-to-instrument studies). Linearity was established across 1.51–15,695 mIU/mL. Interference testing (CLSI EP07) showed no interference (>10% difference) for 40+ substances. Method comparison against the predicate (n=135) yielded a slope of 0.99 and correlation coefficient of 0.998. Matrix comparison (serum vs. Li-Hep/K2-EDTA plasma) showed high correlation (r=0.999).

Technological Characteristics

Sandwich immunoassay; utilizes mouse monoclonal anti-ß-hCG antibody coated on microwells and HRP-labelled mouse monoclonal anti-ß-hCG conjugate. Energy source: VITROS 5600 Integrated System (luminescent detection). Connectivity: Integrated system. Reagents include luminogenic substrates (luminol derivative, peracid salt) and electron transfer agent (substituted acetanilide). Traceable to NIBSC 75/589.

Indications for Use

Indicated for the quantitative measurement of human chorionic gonadotropin (hCG) and its ß-subunit in human serum and plasma (heparin and EDTA) to aid in the early detection of pregnancy in patients.

Regulatory Classification

Identification

A human chorionic gonadotropin (HCG) test system is a device intended for the early detection of pregnancy is intended to measure HCG, a placental hormone, in plasma or urine. A human chorionic goadotropin (HCG) test system is a device intended for any uses other than early detection of pregnancy (such as an aid in the diagnosis, prognosis, and management of treatment of persons with certain tumors or carcinomas) is intended to measure HCG, a placental hormone, in plasma or urine.

Predicate Devices

• VITROS Immunodiagnostic Products Total β-hCG II Reagent Pack (K063720)

Submission Summary (Full Text)

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FDA

U.S. FOOD & DRUG

ADMINISTRATION

# 510(k) SUBSTANTIAL EQUIVALENCE DETERMINATION DECISION SUMMARY

ASSAY ONLY

## I Background Information:

A 510(k) Number

K233581

B Applicant

Ortho Clinical Diagnostics

C Proprietary and Established Names

VITROS Immunodiagnostic Products Total β-hCG II Reagent Pack

D Regulatory Information

| Product Code(s) | Classification | Regulation Section | Panel |
| --- | --- | --- | --- |
| DHA | Class II | 21 CFR 862.1155 - Human Chorionic Gonadotropin (HCG) Test System | CH - Clinical Chemistry |

## II Submission/Device Overview:

A Purpose for Submission:

Modification to a previously cleared device

B Measurand:

Human chorionic gonadotropin

C Type of Test:

Quantitative

## III Intended Use/Indications for Use:

A Intended Use(s):

See Indications for Use below.

Food and Drug Administration

10903 New Hampshire Avenue

Silver Spring, MD 20993-0002

www.fda.gov

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B Indication(s) for Use:

For in vitro diagnostic use only.

For the quantitative measurement of human chorionic gonadotropin (hCG) and its β-subunit in human serum and plasma (heparin and EDTA) using the VITROS 5600 Integrated System to aid in the early detection of pregnancy.

C Special Conditions for Use Statement(s):

Rx - For Prescription Use Only

D Special Instrument Requirements:

VITROS 5600 Integrated System

IV Device/System Characteristics:

A Device Description:

The VITROS Immunodiagnostic Products Total β-hCG II Reagent Pack (test) is performed using the VITROS Immunodiagnostic Products Total β-hCG II Reagent and the VITROS Total β-hCG II Calibrators on the VITROS 5600 Integrated System.

VITROS Immunodiagnostic Products Total β-hCG II Reagent is comprised of the following:

- 100 coated wells (antibody, mouse monoclonal anti-β-hCG, binds >600 mIU hCG/well)
- 14.4 mL assay reagent (buffer containing mouse serum, bovine serum albumin, bovine gamma globulin and antimicrobial agent)
- 19.2 mL conjugate reagent (HRP-mouse monoclonal anti-β-hCG, binds ≥4005 mIU hCG/mL) in buffer with bovine serum albumin and antimicrobial agent

B Principle of Operation:

An immunometric immunoassay technique is used, which involves the reaction of human chorionic gonadotropin (hCG) present in the sample with a microwell coated with biotinylated antibody (mouse monoclonal anti-β-hCG) bound to streptavidin, and a Horseradish Peroxidase (HRP)-labelled antibody conjugate (mouse monoclonal anti-β-hCG). Unbound materials are removed by washing.

The bound HRP-conjugate is measured by a luminescent reaction. A reagent containing luminogenic substrates (a luminol derivative and a peracid salt) and an electron transfer agent, is added to the wells. The HRP in the bound conjugate catalyzes the oxidation of the luminol derivative, producing light. The electron transfer agent (a substituted acetanilide) increases the level of light produced and prolongs its emission. The light signals are read by the system. The amount of HRP conjugate bound is directly proportional to the concentration of hCG present in the sample.

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V Substantial Equivalence Information:

A Predicate Device Name(s):

VITROS Immunodiagnostic Products Total β-hCG II Reagent Pack

B Predicate 510(k) Number(s):

K063720

C Comparison with Predicate(s):

| Device & Predicate Device(s): | K233581 | K063720 |
| --- | --- | --- |
| Device Trade Name | VITROS Immunodiagnostic Products Total β-hCG II Reagent Pack | VITROS Immunodiagnostic Products Total β-hCG II Reagent Pack |
| General Device Characteristic Similarities | | |
| Intended Use/Indications for Use | For the quantitative measurement of human chorionic gonadotropin (hCG) and its β- subunit in human serum and plasma (heparin and EDTA) to aid in the early detection of pregnancy. | Same |
| Sample Type | Serum and Plasma | Same |
| Measuring Range | 2.39– 15,000 mIU/mL | Same |
| Sample Volume | 40 μL | Same |
| Test Principle | Sandwich immunoassay | Same |
| General Device Characteristic Differences | | |
| Biotin-conjugated antibody | Coated onto microwell | In the reagent buffer |

VI Standards/Guidance Documents Referenced:

Clinical and Laboratory Standards Institute (CLSI) EP17-A2, 2013; Evaluation of Detection Capability for Clinical Laboratory Measurement Procedures; Approved Guideline – Second Edition

CLSI EP05-A3, (Reaffirmed: September 2019) Evaluation of Precision Performance of Quantitative Measurement Procedures; Approved Guideline – Third Edition

CLSI EP06: Evaluation of the Linearity of Quantitative Measurement Procedures; Approved Guideline – Second Edition

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CLSI EP07: Interference Testing in Clinical Chemistry; Approved Guideline – Third Edition

CLSI EP37: Supplemental Tables for Interference Testing in Clinical Chemistry – First Edition

CLSI EP35: Assessment of Equivalence or Suitability of Specimen Types for Medical Laboratory Measurement Procedures– First Edition

CLSI EP09c: Measurement Procedure Comparison and Bias Estimation Using Patient Samples – Third Edition

CLSI EP28-A3c: Defining Establishing and Verifying Reference Intervals in the Clinical Laboratory; Approved Guideline - Third Edition

CLSI EP34: Establishing and Verifying an Extended Measuring Interval Through Specimen Dilution and Spiking– First Edition

## VII Performance Characteristics (if/when applicable):

### A Analytical Performance:

#### 1. Precision/Reproducibility:

The precision performance of the device was evaluated using procedures based on the Clinical and Laboratory Standards Institute (CLSI) EP05-A3 guideline.

#### A) 20-Day Precision Study

Six serum pools were prepared by pooling female serum samples to attain the desired concentrations of hCG. Two replicates of each of the six samples were assayed in two runs per day, over 20 days. Three reagent lots and one analyzer were used. Summary data of one representative reagent lot is shown here:

| Sample | Mean (mIU/mL) | Repeatability | | Between-Run | | Between-Day | | Total imprecision | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| | N=80 | SD | %CV | SD | %CV | SD | %CV | SD | %CV |
| Sample 1 | 5.33 | 0.095 | 1.80 | 0.190 | 3.6 | 0.183 | 3.4 | 0.281 | 5.3 |
| Sample 2 | 15.46 | 0.181 | 1.20 | 0.647 | 4.2 | 0.215 | 1.4 | 0.706 | 4.6 |
| Sample 3 | 45.67 | 0.666 | 1.50 | 1.260 | 2.8 | 0.632 | 1.4 | 1.559 | 3.4 |
| Sample 4 | 297.34 | 4.970 | 1.70 | 4.479 | 1.5 | 5.463 | 1.8 | 8.635 | 2.9 |
| Sample 5 | 4708.50 | 82.29 | 1.70 | 95.10 | 2.0 | 83.98 | 1.8 | 151.22 | 3.2 |
| Sample 6 | 9651.00 | 286.71 | 3.00 | 230.87 | 2.4 | 283.26 | 2.9 | 464.48 | 4.8 |

#### B) Instrument-to-Instrument Reproducibility Study

A reproducibility study was conducted to assess instrument-to-instrument variability. Five serum samples with different hCG concentrations were run on three analyzers. The samples

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were tested across each instrument, one lot per instrument, with five replicates per run, one run per day, over five days. The within-run, between-day, between-instrument, and reproducibility was calculated for each reagent lot. Results from multiple lots were similar. Results from one representative lot are provided below:

| Concentration (mIU/mL) | | | Repeatability (Within-run) | | Between-day | | Between - instrument | | Total | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Sample | N | Mean | SD | %CV | SD | %CV | SD | %CV | SD | %CV |
| Sample 1 | 75 | 4.38 | 0.10 | 2.3 | 0.14 | 3.2 | 0.37 | 8.5 | 0.41 | 9.4 |
| Sample 2 | 75 | 15.00 | 0.23 | 1.5 | 0.42 | 2.8 | 0.90 | 6.0 | 1.02 | 6.8 |
| Sample 3 | 75 | 49.55 | 0.77 | 1.6 | 0.94 | 1.9 | 1.96 | 4.0 | 2.31 | 4.7 |
| Sample 4 | 75 | 306.35 | 5.11 | 1.7 | 4.50 | 1.5 | 7.19 | 2.3 | 9.90 | 3.2 |
| Sample 5 | 75 | 4781.1 | 105.94 | 2.2 | 89.54 | 1.9 | 92.27 | 1.9 | 166.59 | 3.5 |
| Sample 6 | 75 | 8329.9 | 249.62 | 3.0 | 219.68 | 2.6 | 220.88 | 2.7 | 399.20 | 4.8 |

## 2. Linearity:

Two linearity studies were performed using 10 serum samples each. Varying concentrations of hCG were obtained by mixing low level serum samples and high level serum samples. Both studies were performed using one lot of reagents on one analyzer.

In the first study, linearity was established by testing samples with concentrations ranging from 38.6-15695 mIU/mL. Each sample was tested in 10 replicates. A regression analysis was conducted resulting in the following linear regression equation:

$$
y = 0.991x + 0.10, R^2 = 0.996
$$

In the second study, linearity was established by testing samples with concentrations ranging from 1.51 to 51.18 mIU/mL. Each sample was tested in 10 replicates. A regression analysis was conducted resulting in the following linear regression equation:

$$
y = 1.025x + 0.07, R^2 = 0.999
$$

The results from the linearity studies support the reportable range of 2.39-15000 mIU/mL (IU/L).

## Dilution studies

Dilution studies were performed to determine the sample recovery after a 1:400 dilution using VITROS High Sample Diluent B performed automatically by the analyzer. The dilution study results support labeling claims that samples with hCG concentrations above 15,000 mIU/mL may be diluted 1:400 using VITROS High Sample Diluent B on-onboard the analyzer.

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# 3. Analytical Specificity/Interference:

The effect of potential interfering substances and cross-reacting substances on the performance of the VITROS Immunodiagnostic Products Total $\beta$ -hCG II Reagent were evaluated based on recommendations in CLSI EP07 Third Edition.

# A) Interference:

Three female human serum pool samples with approximately $5.00\mathrm{mIU / mL}$ (IU/L), 50.00 $\mathrm{mIU / mL}$ (IU/L) and $10,000~\mathrm{mIU / mL}$ (IU/L) ("low", "medium", and "high" respectively) hCG were spiked with various concentrations of potentially interfering substances. Each sample was divided into two aliquots: test (with added interferent) and control (with no added interferent) and measured in replicates of 5 across each of 3 reagent lots on one instrument. No interference ( $>10\%$ difference) was observed with the substances and concentrations listed below.

| Substance | Highest concentration at which no interference was observed |
| --- | --- |
| Acetaminophen | 15.6 mg/dL |
| N-Acetylcysteine | 15.0 mg/dL |
| Alpha-tocopherol | 6.45 mg/dL |
| Amoxicillin | 5.40 mg/dL |
| Ascorbic acid | 5.25 mg/dL |
| Bilirubin, conjugated | 40 mg/dL |
| Bilirubin, unconjugated | 40 mg/dL |
| Biotin | 3510 ng/mL |
| Carbamazepine | 4.50 mg/dL |
| Cefoxitin sodium | 695 mg/dL |
| Cholesterol | 400 mg/dL |
| Codeine | 0.141 mg/dL |
| Colecalciferol | 19.2 μg/dL |
| Cotinine | 0.24 mg/dL |
| Dextran 40 | 2400 mg/dL |
| Dextromethorphan | 0.00156 mg/dL |
| Dipyrone | 100 mg/dL |
| Enoxaparin | 360 U/dL |
| Ethanol | 600 mg/dL |
| Furosemide | 1.59 mg/dL |
| Gamma globulin | 6 g/dL |
| HAMA (Human Anti-Mouse Antibodies) | 800 μg/L |
| Hemoglobin | 1000 mg/dL |
| Hydralazine hydrochloride | 1.44 mg/dL |
| Hydrocodone | 0.0072 mg/dL |
| Ibuprofen | 21.9 mg/dL |
| Intralipid | 2000 mg/dL |
| Levothyroxine | 0.0429 mg/dL |
| Loratadine | 0.0087 mg/dL |

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| Naproxen | 36.0 mg/dL |
| --- | --- |
| Nifedipine | 0.0588 mg/dL |
| Nitrofurantoin | 0.213 mg/dL |
| Omeprazole | 0.840 mg/dL |
| Phenytoin | 6.00 mg/dL |
| Prednisone | 0.010 mg/dL |
| Propranolol HCl | 0.115 mg/dL |
| Rheumatoid factor | 900 IU/mL |
| Salicylic acid | 2.86 mg/dL |
| Sodium azide | 100 mg/dL |
| Sulfamethoxazole | 40 mg/dL |
| Theophylline | 6.0 mg/dL |
| Total Protein | 15 g/dL |
| Triglycerides | 1500 mg/dL |
| Trimethoprim | 4.2 mg/dL |
| Vancomycin hydrochloride | 12.3 mg/dL |

## B) Cross reactivity:

Cross-reactivity of the VITROS Total $\beta$-hCG II reagent pack was determined using female human serum samples with approximately 0 and $25\,\mathrm{mIU/mL}$ hCG concentrations that were spiked with the following potential cross reactants: luteinizing hormone (LH), follicle stimulating hormone (FSH), or thyroid stimulating hormone (TSH). Samples were tested in replicates of five using 3 lots of reagents on one analyzer.

Cross-reactivity at the $0\,\mathrm{mIU/mL}$ hCG concentration was calculated using the following equation:

$$
\%$ cross-reactivity = $\frac{\text{Mean result of cross-reactant sample} \times 100}{\text{Concentration of cross-reactant}}$

Cross-reactivity at the $25\,\mathrm{mIU/mL}$ hCG concentration was calculated using the following equation:

$$
\%$ cross-reactivity = $\frac{(\text{Mean result of cross-reactant sample} - \text{Mean result of control sample}) \times 100}{\text{Concentration of cross-reactant}}
$$

No cross-reactivity at the tested concentrations was detected at $0\,\mathrm{mIU/mL}$ hCG and the cross-reactivity at the tested concentrations observed at $25\,\mathrm{mIU/mL}$ hCG is listed in the table below:

| Substance | Tested Concentration | VITROS 5600 - % Cross Reactivity | | |
| --- | --- | --- | --- | --- |
| | | Lot 1 | Lot 2 | Lot 3 |
| FSH | 400 mIU/mL | 0.6% | 0.3% | 0.3% |
| LH | 400 mIU/mL | -0.3% | -0.1% | -0.5% |
| TSH | 200 mIU/mL | 0.4% | 0.1% | -0.3% |

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4. Assay Reportable Range:

The assay reportable range is 2.39 to 15,000 mIU/mL. See section VII.A.2 - Linearity and section VII.A.6 - Detection Limit.

5. Traceability, Stability, Expected Values (Controls, Calibrators, or Methods):

The VITROS Total β-hCG II Reagent Pack is traceable to the World Health Organization (WHO) 4th International Standard Chorionic Gonadotropin, Human, National Institute for Biological Standards and Control (NIBSC), code 75/589.

6. Detection Limit:

The Limit of Blank (LoB), Limit of Detection (LoD) and Limit of Quantitation (LoQ) for the VITROS Immunodiagnostic Products Total β-hCG II Reagent Pack were determined using procedures based on the CLSI EP17-A2 guideline.

For determination of the LoB, four analyte-free serum samples were tested in replicates of five per run, with three lots of reagent, on one analyzer, over five days. The LoB was determined to be 0.00 mIU/mL. The results supports the claimed LoB of 0.05 mIU/mL.

For determination of the LoD, five serum samples containing low analyte concentrations (approximately 1 – 5 times LoB) were tested in replicates of 12 per day, using three reagent lots, on one analyzer over 5 calendar days. The LoD was determined to be 0.21 mIU/mL. The results support the claimed LoD of 0.70 mIU/mL.

For determination of the LoQ, four serum sample pools that contained low analyte concentrations were used to evaluate the LoQ using the Total Error (<20%) approach. The LoQ using the Total Error (<20%) approach was calculated to be 2.32 mIU/mL. The results support the claimed LoQ of 2.39 mIU/mL.

7. Assay Cut-Off:

Not applicable.

B Comparison Studies:

1. Method Comparison with Predicate Device:

Method comparison studies were conducted following recommendations in the CLSI EP09c-3rd Edition guideline.

A total of 135 serum samples were measured in singlicate using the updated VITROS Immunodiagnostic Products Total β-hCG II Reagent Pack (candidate device) and the predicate device. The samples were tested using one reagent lot and one analyzer. The results were assessed using Weighted Deming regression analysis and the results are shown in the following table:

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| n | Slope (95% CI) | Correlation Coefficient | Conventional Units (mIU/mL) | |
| --- | --- | --- | --- | --- |
| | | | Range of Samples | Intercept (95% CI) |
| 135 | 0.99 (0.977 to 0.995) | 0.998 | 2.78 to 14,675 | -0.0215 (-0.160 to 0.117) |

2. Matrix Comparison:

Performance of the VITROS Immunodiagnostic Products Total $\beta$-hCG II Reagent with different sample matrices was evaluated based on the recommendations in the CLSI EP35 guideline.

A matrix comparison study was performed to evaluate matrices suitable for use with the VITROS Immunodiagnostic Products Total $\beta$-hCG II Reagent Pack. Serum samples with concentrations ranging from 7.55- 10580.0 mIU/mL were compared to matched Lithium Heparin and K2-EDTA plasma samples using one lot of reagents on the analyzer. The results were assessed using Weighted Deming regressions analysis.

A summary of the results, when compared to serum, are shown in table below:

| Parameter | Li-Hep Plasma | K2-EDTA Plasma |
| --- | --- | --- |
| Slope | 0.978 | 0.978 |
| Intercept | 0.1708 | 0.0529 |
| Correlation Coefficient r | 0.999 | 0.999 |
| n | 41 | 41 |

C Clinical Studies:

1. Clinical Sensitivity:

Not applicable.

2. Clinical Specificity:

Not applicable.

3. Other Clinical Supportive Data (When 1. and 2. Are Not Applicable):

Not applicable.

D Clinical Cut-Off:

Not applicable.

E Expected Values/Reference Range:

The reference range information was reviewed in K063720.

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VIII Proposed Labeling:

The labeling supports the finding of substantial equivalence for this device.

IX Conclusion:

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

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