The BioPlex 2200 EBV IgM kit is a multiplex flow immunoassay intended for the qualitative detection of two (2) separate analytes; Epstein-Barr Virus Viral Capsid Antigen (EBV VCA) IgM antibodies and Heterophile antibodies in human serum. The test system can be used in conjunction with the BioPlex 2200 EBV IgG kit as an aid in the laboratory diagnosis of infectious mononucleosis (IM).

The EBV IgM kit is intended for use with the Bio-Rad BioPlex 2200 System.

Assay performance characteristics have not been established for immunocompromised or immunosuppressed patients, cord blood, neonatal specimens, or infants. Assay performance characteristics have not been established for the diagnosis of nasopharyngeal carcinoma, Burkitt's lymphoma, and other EBV-associated lymphomas.

BioPlex 2200 EBV IgM Calibrator Set: The BioPlex 2200 EBV IgM Calibrator Set is intended for the calibration of the BioPlex 2200 EBV IgM Reagent Pack.

BioPlex 2200 EBV IgM Control Set: The BioPlex 2200 EBV IgM Control Set is intended for use as an assayed quality control to monitor the overall performance of the BioPlex 2200 Instrument and BioPlex 2200 EBV IgM Reagent Pack in the clinical laboratory. The performance of the BioPlex 2200 EBV IgM Control Set has not been established with any other EBV assays.

Device Description

The BioPlex 2200 EBV IgM kit uses multiplex flow immunoassay, a methodology that greatly resembles traditional ElA, but permits simultaneous detection and identification of many antibodies in a single tube. Two (2) different populations of dyed beads are coated with proteins associated with infectious mononucleosis. One (1) is coated with an E. coli derived recombinant fusion protein, EBV VCA p18 (40kD), and the other is coated with horse erythrocyte stromal extract (heterophile antigen). The BioPlex 2200 System combines an aliquot of patient sample, sample diluent containing goat anti-human IgG, and bead reagent into a reaction vessel. The mixture is incubated at 37°C. After a wash cycle, anti-human IgM antibody, conjugated to phycoerythrin (PE), is added to the dyed beads and this mixture is incubated at 37°C. The excess coniugate is removed in another wash cycle, and the beads are re-suspended in wash buffer. The bead mixture then passes through the detector. The identity of the dyed beads is determined by the fluorescence of the dyes, and the amount of antibody captured by the antigen is determined by the fluorescence of the attached PE.

Three additional dyed beads, an Internal Standard Bead (ISB), a Serum Verification Bead (SVB), and a Reagent Blank Bead (RBB), are present in each reaction mixture to verify detector response, the addition of serum or plasma to the reaction vessel, and the absence of significant non-specific binding in serum or plasma respectively. The instrument is calibrated using a set of two (2) distinct calibrator vials, supplied separately by Bio-Rad Laboratories. A combination of two (2) vials representing two (2) different antibody concentrations is used for calibration. The result for each of these antibodies is expressed as an antibody index (AI).

AI/ML Overview

Here's a summary of the acceptance criteria and study details for the BioPlex 2200 EBV IgM Kit, Calibrators, and Controls, based on the provided text:

1. Table of Acceptance Criteria and Reported Device Performance

The document does not explicitly present a table of acceptance criteria in numerical targets for performance metrics like sensitivity, specificity, or %CV. Instead, it presents the results of the studies conducted and implies that these results were deemed acceptable for FDA clearance. The performance is assessed through reproducibility, precision, and comparative testing against predicate devices and known serological patterns.

However, we can infer performance metrics from the results provided:

Performance Metric	Acceptance Criteria (Implied)	Reported Device Performance (Summary from text)
Reproducibility (EBV VCA IgM)	Low %CV for within-run, between-day, between-run, between-site.	High Positive 1 (AI 1.9): Total %CV 8.4% High Positive 2 (AI 2.0): Total %CV 7.1% Low Positive 1 (AI 1.2): Total %CV 8.3% Low Positive 2 (AI 1.4): Total %CV 7.9% Positive Control (AI 2.0): Total %CV 17.2%
Reproducibility (Heterophile)	Low %CV for within-run, between-day, between-run, between-site.	High Positive 1 (AI 2.8): Total %CV 7.8% High Positive 2 (AI 2.7): Total %CV 5.5% Low Positive 1 (AI 1.9): Total %CV 5.5% Low Positive 2 (AI 1.8): Total %CV 7.4% Positive Control (AI 2.5): Total %CV 5.9%
Precision (EBV VCA IgM)	Low %CV for within-run, between-day, between-run.	High Positive 1 (AI 2.5): Total %CV 11.2% High Positive 2 (AI 2.7): Total %CV 9.1% Low Positive 1 (AI 1.6): Total %CV 7.8% Low Positive 2 (AI 1.9): Total %CV 7.1% High Negative (AI 0.7): Total %CV 7.0% Low Negative (AI 0.1): Total %CV 0.0%
Precision (Heterophile)	Low %CV for within-run, between-day, between-run.	High Positive 1 (AI 2.8): Total %CV 9.5% High Positive 2 (AI 2.8): Total %CV 12.2% Low Positive 1 (AI 2.1): Total %CV 10.5% Low Positive 2 (AI 1.9): Total %CV 9.6% High Negative (AI 0.7): Total %CV 8.4% Low Negative (AI 0.0): Total %CV 0.0%
Comparative Testing (EBV VCA IgM vs. EIA) Percent Agreement by Serological Pattern Characterization (Table 18)	High positive and negative agreement.	Primary Acute: Positive 96.8% (95% CI 83.8-99.4%) Late Acute: Positive 64.6% (95% CI 50.4-76.6%), Negative 91.9% (95% CI 82.5-96.5%) Recovering: Negative 75.0% (95% CI 30.1-95.4%) Previous Infection: Negative 96.1% (95% CI 93.2-97.7%) Susceptible: Negative 96.9% (95% CI 92.2-98.8%) Inconclusive: Positive 100% (95% CI 51.0-100%), Negative 83.8% (95% CI 68.9-92.3%) Overall: Positive 78.3% (95% CI 68.3-85.8%), Negative 94.8% (95% CI 92.5-96.4%)
Comparative Testing (Heterophile vs. Agglutination Test) Percent Agreement by Serological Pattern Characterization (Table 20)	High positive and negative agreement.	Primary Acute: Positive 84.2% (95% CI 62.4-94.5%), Negative 83.3% (95% CI 55.2-95.3%) Late Acute: Positive 75.0% (95% CI 30.1-95.4%), Negative 98.1% (95% CI 93.4-99.5%) Recovering: Negative 100% (95% CI 51.0-100%) Previous Infection: Negative 99.0% (95% CI 97.1-99.7%) Susceptible: Negative 100% (95% CI 97.1-100%) Inconclusive: Positive 14.8% (95% CI 5.9-32.5%), Negative 100% (95% CI 78.5-100%) Overall: Positive 46.0% (95% CI 33.0-59.6%), Negative 98.8% (95% CI 97.5-99.4%)
Overall Serological Agreement (Table 25)	High overall serological agreement.	Primary Acute: 96.8% (95% CI 83.8-99.4%) Late Acute: 81.8% (95% CI 73.6-87.9%) Recovering: 75.0% (95% CI 30.0-95.4%) Previous Infection: 86.2% (95% CI 81.9-89.7%) Susceptible: 96.1% (95% CI 91.1-98.3%) Inconclusive: 17.1% (95% CI 8.5-31.3%) Overall: 83.3% (95% CI 80.2-86.1%)
Acute vs. Non-acute Serological Agreement (Table 26)	High agreement for acute and non-acute classifications.	Acute: 88.7% (95% CI 82.4-92.9%) Non-Acute: 90.4% (95% CI 87.2-92.8%) Inconclusive: 17.1% (95% CI 8.5-31.3%) Overall: 85.1% (95% CI 82.1-87.7%)
Cross-Reactivity	Minimal or no cross-reactivity with tested interfering factors.	Generally low cross-reactivity. A few discrepancies were noted for Toxoplasmosis and CMV IgM samples (up to 5 and 3 discrepants respectively for EBV VCA IgM, and 1 for Heterophile with CMV IgM). Most positive BioPlex 2200 results in these categories were confirmed by commercial assays.

2. Sample Size and Data Provenance

Test Set (Comparative Testing):
- Main Comparative Study: 621 banked serum samples from patients for whom an EBV test was ordered. Two samples were excluded due to RBB errors, resulting in N=619 for initial analyses and N=618 for serological pattern comparisons.
  - Provenance: 3 U.S. clinical testing sites (retrospective, banked serum samples).
- Known EBV VCA IgM Positive Samples: 100 purchased EBV VCA IgM positive samples.
  - Provenance: A U.S. clinical testing site (retrospective, purchased samples).
Reproducibility and Precision Studies: These studies used "panel members" prepared by Bio-Rad Laboratories. The number of panel members was 6 (2 high positive, 2 near cutoff, 2 negative). Each panel member was tested multiple times across days and sites. These are typically internal validation samples, not patient data in the same way as the comparative testing.

3. Number of Experts and Qualifications

The document does not specify the number or qualifications of experts used to establish the ground truth for the test set. The ground truth in comparative testing was established by "corresponding commercially available microplate EIA/agglutination tests" and subsequent serological pattern characterization. It is implied that these predicate devices and the serological algorithm (Table 16) serve as the 'expert' reference. For the purpose of result interpretation, the algorithm defines different EBV serological statuses based on various antibody responses.

4. Adjudication Method

The document does not describe an adjudication method for the test set by human experts in the traditional sense (e.g., 2+1, 3+1). Instead, the results of the BioPlex 2200 system were compared to the results of predicate commercial assays. For percent agreement calculations in comparative testing, BioPlex 2200 "equivocal results were assigned to the opposite clinical interpretation than that of the corresponding reference assay result." This acts as a conservative rule for calculating agreement.

5. Multi-Reader Multi-Case (MRMC) Comparative Effectiveness Study

No MRMC comparative effectiveness study was described. The device is an automated in vitro diagnostic (IVD) device for detecting antibodies, not an imaging device requiring human reader interpretation. Therefore, a study to measure human reader improvement with AI assistance is not applicable. The comparison is between the new automated assay and existing commercial laboratory assays (EIA and agglutination tests).

6. Standalone Performance

Yes, a standalone performance study was done. The reproducibility and precision studies evaluate the device's inherent performance characteristics as a standalone assay. The comparative testing also assesses the BioPlex 2200 EBV IgM kit alone against predicate devices, indicating its standalone diagnostic utility. The results (Antibody Index (AI) values, %CV, positive/negative agreement with reference methods) are presented for the BioPlex 2200 system directly.

7. Type of Ground Truth Used

The ground truth used for the comparative studies was:

Reference Assays: Results from "corresponding commercially available microplate EIA/agglutination tests." These are established diagnostic assays for EBV.
Serological Pattern Characterization: A generally accepted algorithm for classifying patients into EBV serological status (Table 16) based on results from various EBV antibody tests (including EBV NA-1 IgG, EBV VCA IgG, EBV EA-D IgG, EBV VCA IgM, and Heterophile Antibody).

8. Sample Size for the Training Set

The document does not explicitly mention a "training set" in the context of an AI/ML algorithm, as this device appears to be a laboratory immunoassay rather than an AI-driven one. Therefore, there is no concept of a training set for an algorithm in this submission. The panel members for reproducibility and precision studies can be thought of as internal validation sets, but not "training sets" for an AI model.

9. How the Ground Truth for the Training Set was Established

As there is no mention of an AI/ML model or a specific training set with ground truth in the document, this question is not applicable. The characterization of panel members for reproducibility and precision studies would have been established internally by Bio-Rad Laboratories based on known antibody levels or clinical samples.

Summary

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KOC2213

DEC - 8 2006

BIOPLEX 2200 EBV IgM KIT, CALIBRATORS, AND CONTROLS 510(k) SUMMARY

This summary of 510(k) safety and effectiveness information is being submitted in accordance with the requirements of SMDA 1990 and 21 CFR 807.92.

510(k) Number	510(k) Summary Report Date
K062213	December 7, 2006

MANUFACTURER INFORMATION

Manufacturer
Manufacturer Address	Bio-Rad Laboratories, Inc.Clinical Systems Division4000 Alfred Nobel DriveHercules, CA 94547
Telephone	(510) 724-7000
Establishment Registration No.	2915274
Owner / Operator	Bio-Rad Laboratories, Inc.4000 Alfred Nobel DriveHercules, CA 94547
Owner / Operator No.	9929003
	Official Correspondent for the BioPlex 2200 EBV IgM
Official Correspondent Address	Bio-Rad Laboratories6565 185th Ave NERedmond, WA 98052
Telephone	425-881-8300
Establishment Registration No.	3022521
Owner / Operator	Bio-Rad Laboratories6565 185th Ave NERedmond, WA 98052
Official Correspondent	Mr. Christopher Bentsen
Telephone	(425) 498-1709
Fax	(425) 498-1651

CLASSIFICATION INFORMATION

Classification Name	Epstein Barr Virus, Other (LSE)
Common Name:	Multi-Analyte Detection System EBV IgM
Product Trade Name	BioPlex 2200 EBV IgM on the BioPlex 2200 Multi-AnalyteDetection SystemBioPlex 2200 EBV IgG Control SetBioPlex 2200 EBV IgG Calibrator Set
Device Class	Class I
Classification Panel	Microbiology
Regulation Number	866.3235

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	BioPlex2200 EBVIgG Analyte	Comparative FDA ClearedPREDICATE DEVICE	510(k)Number	DecisionDate
1.	EBV VCA	Diasorin EBV VCA IgM Capture EIA	946157	6/7/95
2.	Heterophile	Wampole Monolatex	862008	7/03/86

LEGALLY MARKETED EQUIVALENT (SE) DEVICES

DEVICE DESCRIPTION

KIT COMPONENTS

EBV IgM Reagent Pack (Catalog No. 665-1350). The reagent pack contains supplies sufficient for 100 tests.

Vial	Description
Bead Set	One (1) 10 mL vial, containing 2 different populations of dyed beads coatedwith affinity-purified E. coli derived recombinant protein to EBV VCA p18(40kD), and Heterophile antigen (horse erythrocyte stromal extract); anInternal Standard (ISB), a Serum Verification (SVB), and a Reagent Blank(RBB); with Glycerol and protein stabilizers (bovine) in a MOPS (3-[N-Morpholino] propanesulfonic acid) buffer. ProClin® 300 (0.3%) and sodiumazide (<0.1%) as preservatives.
Conjugate	One (1) 5 mL vial, containing donkey anti-human IgM/phycoerythrinconjugate and murine monoclonal anti-human FXIII / phycoerythrinconjugate, in a phosphate buffer. Proclin® 300 (0.3%) and Sodium azide(0.1%) as preservatives.
Sample Diluent	One (1) 10 mL vial, containing goat anti-human IgG and protein stabilizers(bovine and murine) in a triethanolamine buffer. Proclin® 300 (0.3%) andSodium azide (0.1%) as preservatives.

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ADDITIONAL REQUIRED ITEMS, AVAILABLE FROM BIO-RAD

Catalog #	Description
663-1300	BioPlex 2200 EBV IgM Calibrator Set: Two (2) 500 µL vials, containing EBVVCA and heterophile antibodies, in a human serum matrix made fromdefibrinated plasma. ProClin® 300 (0.3%) as a preservative for allcalibrators.
663-1330	BioPlex 2200 EBV IgM Control Set: Two (2) 1.5 mL vials of Positive Controlcontaining EBV VCA and heterophile antibodies, in a human serum matrixmade from defibrinated plasma; and two (2) 1.5 mL vials of NegativeControl in a human serum matrix made from defibrinated plasma. ProClin®300 (0.3%) as a preservative for all controls.
660-0817	BioPlex 2200 System Sheath Fluid: Two (2) 4 L bottles containingPhosphate Buffered Saline (PBS). Proclin® 300 (0.3%) and Sodium azide(0.1%) as preservatives.
660-0818	BioPlex 2200 System Wash Solution: One (1) 10 L bottle containingPhosphate Buffered Saline (PBS) and Tween 20. Proclin® 300 (0.3%) andSodium azide (0.1%) as preservatives.
660-0000	BioPlex 2200 Instrument and Software.

INTENDED USE / INDICATIONS FOR USE

The EBV IqM kit is intended for use with the Bio-Rad BioPlex 2200 System.

BioPlex 2200 EBV IgM Calibrator Set

The BioPlex 2200 EBV IgM Calibrator Set is intended for the calibration of the BioPlex 2200 EBV IgM Reagent Pack.

BioPlex 2200 EBV IgM Control Set

The BioPlex 2200 EBV IgM Control Set is intended for use as an assayed quality control to monitor the overall performance of the BioPlex 2200 Instrument and BioPlex 2200 EBV IgM Reagent Pack in the clinical laboratory. The performance of the BioPlex 2200 EBV IgM Control Set has not been established with any other EBV assays.

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TECHNOLOGICAL CHARACTERISTICS

The following tables summarize similarities and differences between the BioPlex 2200 EBV IgG Kit, Calibrators, and Controls and the predicate devices used in comparative studies with the BioPlex 2200 EBV IgG Kit.

A. BioPlex 2200 EBV IgM Assay: EBV VCA

		Table 1: Similarities between reagents and materials

Similarities betweenComponents /Materials	BioPlex 2200 EBV IgM Kit	Predicate EBV VCA IgMReverse Capture EIA
Reagents	Wash Buffer, Sample Diluent	Wash Buffer, Sample Diluent
Calibrator(s)	Calibrators	Calibrators
Controls	Negative Control and Multi-Analyte Positive Control (EBVVCA, EBV NA-1, and EBV EA-D)	Negative Control, Low PositiveControl, and High PositiveControl

Table 2: Similarities between reagents with regard to function and use

Similarities betweenFunction and Use	BioPlex 2200 EBV IgM Kit	Predicate EBV VCA IgMReverse Capture EIA
Intended Use	Qualitative detection of IgMantibodies in human serum toEBV Viral Capsid Antigen (VCA).	Qualitative determination of IgMantibodies in human serum toEBV Viral Capsid Antigen (VCA).
Matrices	Serum	Serum

Table 3: Differences between reagents and materials

Differences betweenComponents /Materials	BioPlex 2200 EBV IgM Kit	Predicate EBV VCA IgMReverse Capture EIA
Solid Phase	Bead reagent - dyed antigencoated beads	96 well microplate - antigencoated microwells
Reagents	Conjugate (Anti-human IgM /Phycoerythrin)	Enzyme Tracer (Rat monoclonalanti-p18 conjugated with horse-radish peroxidase), TracerDiluent, Reconstitution Solution,Chromogen / Substrate (TMB),Stop Solution
Sheath Fluid	Sheath Fluid is used to suspendthe bead reagent and introduce itinto the detector.	Not similar; not utilized in EIA's.

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Differences betweenFunction and Use	BioPlex 2200 EBV IgM Kit	Predicate EBV VCA IgMReverse Capture ElA
Intended Use	Qualitative detection of IgMantibodies in human serum toEBV Viral Capsid Antigen (VCA).	Semi-quantitative detection ofIgM antibodies in human serumto EBV Viral Capsid Antigen(VCA).
Analyte Detection	Multi-Analyte Detection (humanIgG antibodies to EBV VCA, EBVNA-1, and EBV EA-D)	Single Analyte Detection(human IgM antibody to EBVViral Capsid Antigen)

Table 4: Differences between reagents with regard to function and use

B. BioPlex 2200 EBV IgM Assay: Heterophile

Table 5: Similarities between reagents and materials

Similarities betweenComponents /Materials	BioPlex 2200 EBV IgM Kit	Predicate MONO-LATEX LatexAgglutination Test
Controls	Negative Control and Multi-Analyte Positive Control (EBVVCA IgM and Heterophile)	Negative Control and PositiveControl

Table 6: Similarities between reagents with regard to function and use

Similarities betweenComponents /Materials	BioPlex 2200 EBV IgM Kit	Predicate MONO-LATEX LatexAgglutination Test
Intended Use	Qualitative detection ofheterophile antibodies	Qualitative detection ofheterophile antibodies
Matrices	Serum	Serum

Table 7: Differences between reagents and materials

Differences betweenComponents /Materials	BioPlex 2200 EBV IgM Kit	Predicate MONO-LATEX LatexAgglutination Test
Solid Phase	Bead reagent - dyed antigencoated beads	Latex reagent - suspension ofmononucleosis antigensensitized latex particles
Reagents	Sample Diluent, Conjugate, WashBuffer, Sheath Fluid	Not similar; these reagents notutilized in latex agglutinationtests.
Calibrators	Calibrators	Not similar; not utilized in latexagglutination tests.

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Differences betweenComponents /Materials	BioPlex 2200 EBV IgM Kit	Predicate MONO-LATEX LatexAgglutination Test
Intended Use	Qualitative detection ofheterophile antibodies.	Semi-quantitative detection ofheterophile antibodies.
Matrices	Serum	Plasma
Analyte Detection	Multi-Analyte Detection (humanEBV VCA IgM and heterophileantibodies)	Single Analyte Detection(human heterophile antibodies)

Table 8: Differences between reagents with regard to function and use

PERFORMANCE SUMMARY

A. Expected Values

Expected values for the EBV IgM kit are presented by age and gender in the following tables for serum samples from unselected hospitalized pediatric and adult patients (N=302) and patients for which an EBV test was ordered (N=619). A total of 303 serum samples from unselected hospitalized pediatric and adult patients and a total of 621 serum samples from patients for which an EBV test was ordered were tested. One (1) sample from the unselected hospitalized population, and two (2) samples from the patients for which an EBV test was ordered population were excluded due to RBB analysis error messages during BioPlex 2200 EBV IgM testing. For all analytes, results of ≤0.8 Al are negative, 0.9 and 1.0 Al are equivocal, and ≥1.1 Al are reported as positive.

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Age	Gender	BioPlex 2200 EBV VCA IgM		Total
		Positive		Equivocal		Negative
		N	%	N	%	N	%	N
< 5 years of age	F	3	11%	0	0%	24	89%	27
	M	1	5%	0	0%	19	95%	20
5-12 years of age	F	2	9%	0	0%	20	91%	22
	M	1	3%	1	3%	32	94%	34
13-20 years of age	F	5	14%	1	3%	29	83%	35
	M	0	0%	1	7%	14	93%	15
21-30 years of age	F	0	0%	0	0%	6	100%	6
	M	0	0%	0	0%	2	100%	2
31-40 years of age	F	0	0%	0	0%	10	100%	10
	M	0	0%	0	0%	11	100%	11
41-50 years of age	F	1	8%	0	0%	12	92%	13
	M	1	14%	0	0%	6	86%	7
51-60 years of age	F	0	0%	0	0%	23	100%	23
	M	0	0%	1	5%	18	95%	19
61-70 years of age	F	0	0%	0	0%	11	100%	11
	M	0	0%	0	0%	11	100%	11
71-80 years of age	F	1	9%	0	0%	10	91%	11
	M	0	0%	0	0%	6	100%	6
81-90 years of age	F	0	0%	0	0%	11	100%	11
	M	0	0%	0	0%	6	100%	6
91-100 years of age	F	0	0%	0	0%	0	0%	0
	M	0	0%	0	0%	2	100%	2
Total		15	5%	4	1%	283	94%	302

Table 9: Hospitalized Patient Samples: EBV VCA IgM

Table 10: Hospitalized Patient Samples: Heterophile
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Age	Gender	Positive			Negative		Total
< 5 years of age	F	1	4%	0%	26	96%	27
< 5 years of age	M	0	0%	0%	20	100%	20
5-12 years of age	F	0	0%	0%	22	100%	22
5-12 years of age	M	1	3%	0%	33	97%	34
13-20 years of age	F	0	0%	0%	35	100%	35
13-20 years of age	M	0	0%	0%	15	100%	15
21-30 years of age	F	0	0%	0%	6	100%	6
21-30 years of age	M	0	0%	0%	2	100%	2
31-40 years of age	F	0	0%	0%	10	100%	10
31-40 years of age	M	0	0%	0%	11	100%	11
41-50 years of age	F	0	0%	0%	13	100%	13
41-50 years of age	M	0	0%	0%	7	100%	7
51-60 years of age	F	0	0%	0%	23	100%	23
51-60 years of age	M	0	0%	0%	19	100%	19
61-70 years of age	F	0	0%	0%	11	100%	11
61-70 years of age	M	0	0%	0%	11	100%	11
71-80 years of age	F	0	0%	0%	11	100%	11
71-80 years of age	M	0	0%	0%	6	100%	6
81-90 years of age	F	0	0%	0%	11	100%	11
81-90 years of age	M	0	0%	0%	6	100%	6
91-100 years of age	F	0	0%	0%	0	0%	0
91-100 years of age	M	0	0%	0%	2	100%	2
Total		2	1%	0%	300	99%	302

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Age	Gender	Positive		Equivocal		Negative		Total
		N	%	N	%	N	%	N
< 5 years of age	F	3	10%	0	0%	27	90%	30
< 5 years of age	M	6	18%	0	0%	27	82%	33
5-12 years of age	F	9	15%	1	2%	52	84%	62
5-12 years of age	M	6	10%	1	2%	55	89%	62
13-20 years of age	F	17	22%	2	3%	59	76%	78
13-20 years of age	M	10	26%	1	3%	27	71%	38
21-30 years of age	F	5	11%	1	2%	40	87%	46
21-30 years of age	M	7	21%	1	3%	25	76%	33
31-40 years of age	F	5	10%	1	2%	46	88%	52
31-40 years of age	M	1	4%	0	0%	23	96%	24
41-50 years of age	F	3	9%	1	3%	30	88%	34
41-50 years of age	M	4	13%	1	3%	26	84%	31
51-60 years of age	F	4	15%	1	4%	22	81%	27
51-60 years of age	M	2	8%	1	4%	23	88%	26
61-70 years of age	F	1	8%	0	0%	12	92%	13
61-70 years of age	M	0	0%	0	0%	21	100%	21
71-80 years of age	F	0	0%	0	0%	2	100%	2
71-80 years of age	M	0	0%	0	0%	3	100%	3
81-90 years of age	F	0	0%	0	0%	2	100%	2
81-90 years of age	M	0	0%	0	0%	2	100%	2
91-100 years of age	F	0	0%	0	0%	0	0%	0
91-100 years of age	M	0	0%	0	0%	0	0%	0
Total		83	13%	12	2%	524	85%	619

Table 11: Samples from Patients for which an EBV Test was Ordered: EBV VCA IgM

										Table 12: Samples from Patients for which an EBV Test was Ordered: Heterophile
--	--	--	--	--	--	--	--	--	--	--------------------------------------------------------------------------------

Age	Gender	Positive		Equivocal		Negative		Total
		N	%	N	%	N	%	N
< 5 years of age	F	0	0%	0	0%	30	100%	30
< 5 years of age	M	0	0%	0	0%	33	100%	33
5-12 years of age	F	3	5%	0	0%	59	95%	62
5-12 years of age	M	3	5%	0	0%	59	95%	62
13-20 years of age	F	8	10%	1	1%	69	88%	78
13-20 years of age	M	7	18%	1	3%	30	79%	38
21-30 years of age	F	0	0%	2	4%	44	96%	46
21-30 years of age	M	4	12%	0	0%	29	88%	33
31-40 years of age	F	0	0%	0	0%	52	100%	52
31-40 years of age	M	1	4%	1	4%	22	92%	24
41-50 years of age	F	0	0%	0	0%	34	100%	34
41-50 years of age	M	0	0%	0	0%	31	100%	31
51-60 years of age	F	0	0%	0	0%	27	100%	27
51-60 years of age	M	0	0%	0	0%	26	100%	26
61-70 years of age	F	0	0%	0	0%	13	100%	13
61-70 years of age	M	0	0%	0	0%	21	100%	21
71-80 years of age	F	0	0%	0	0%	2	100%	2
71-80 years of age	M	0	0%	0	0%	3	100%	3
81-90 years of age	F	0	0%	0	0%	2	100%	2
81-90 years of age	M	0	0%	0	0%	2	100%	2
91-100 years of age	F	0	0%	0	0%	0	0%	0
91-100 years of age	M	0	0%	0	0%	0	0%	0
Total		26	4%	5	1%	588	95%	619

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Image /page/8/Picture/0 description: The image shows the logo for BIO-RAD. The logo is white text on a black background. The text is in all caps and bolded.

The distribution of BioPlex 2200 EBV VCA IgM and Heterophile Al values for serum samples from adult and pediatric unselected hospitalized patients and from patients for which an EBV test was ordered are presented in the following histograms.

Image /page/8/Figure/2 description: Figure 1 shows hospitalized patient samples of EBV VCA IgM using BioPlex 2200 VCA IgM. The x-axis is the antibody index (AI) and the y-axis is the number of patients. For adults, the number of patients is highest at 0.2 AI with a value of 125, while for children, the number of patients is highest at 0.2 AI with a value of 120.

Figure 3. Samples from Patients for which an EBV Test was Ordered: EBV VCA IgM

Image /page/8/Figure/4 description: The image is a bar graph titled "BioPlex 2200 VCA IgM". The x-axis is labeled "Antibody Index (AI)" and ranges from 0.2 to 4.0. The y-axis is labeled "Number of Patients" and ranges from 0 to 250. The graph compares the number of adults and children at each antibody index.

Image /page/8/Figure/5 description: The image is a bar graph titled "Figure 2: Hospitalized Patient Samples: Heterophile BioPlex 2200 Heterophile". The y-axis is labeled "Number of Patients", and the x-axis is labeled "Antibody Index (AI)". The bar graph compares the number of adults and children at different antibody index levels. At an antibody index of 0.2, there are approximately 150 adults and 149 children.

Figure 4. Samples from Patients for which an EBV Test was Ordered: Heterophile

Image /page/8/Figure/7 description: This image is a bar graph titled "BioPlex 2200 Heterophile". The x-axis is labeled "Antibody Index (AI)" and ranges from 0.2 to 4.0. The y-axis is labeled "Number of Patients" and ranges from 0 to 350. The graph compares the number of adults and children at each antibody index, with the highest number of adults being 296 and children being 273 at 0.2 antibody index.

B. Reproducibility Studies

A reproducibility panel, consisting of six (6) panel members was prepared by Bio-Rad Laboratories. Two (2) of the six (6) panel members had high levels of the antibodies contained in the BioPlex 2200 EBV IgM kit (EBV VCA IgM and Heterophile) and two (2) of the six (6) panel members had antibody levels near the cutoff, both prepared from positive patient samples. Two (2) of the six (6) panel members were negative (one high negative and one low negative) for both of the analytes. In addition, a positive control (antibody positive for both analytes) and a negative control (antibody negative for both analytes) were also tested. Reproducibility testing was performed at each of three (3) US testing facilities on a total of three (3) lots of the EBV IgM kit, three (3) lots of the EBV IgM Calibrator Set and three (3) lots of the EBV IgM Control Set. The panels were provided to each of the testing sites. Each of the six (6) panel members and positive and negative controls was tested in quadruple (x4) on each day for three (3) days at each of three (3) US testing facilities using one (1) lot of EBV IgM kit, one (1) lot of EBV IgM Calibrator Set and one (1) lot of EBV IgM Control Set (4 times x 3 days x 3 sites = 36 replicates per panel member and controls). The data were analyzed for intra-assay and inter-assay reproducibility according to the principles described in the Clinical Laboratory Standards Institute guidance EP5-A2, revised November 2004 and ISO/TR 22971:2205. The standard deviation (SD) and percent coefficient of variation (%CV) were calculated. Positive results can be found in Tables 12 and 13.

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Image /page/9/Picture/0 description: The image shows the logo for Bio-Rad Laboratories. The logo is white text on a black, rounded rectangle. The text is in a bold, sans-serif font.

EBV VCA IgMPanel Members	SampleN	GrandMeanAl	Within-Run		Between-Day		Between-Run		Between-Site*		Total
			SD	%CV	SD	%CV	SD	%CV	SD	%CV	SD	%CV
High Positive 1	36	1.9	0.1	5.9%	0.1	4.5%	0.1	3.8%	0.0	0.0%	0.2	8.4%
High Positive 2	36	2.0	0.1	3.5%	0.0	0.0%	0.1	5.8%	0.0	2.2%	0.1	7.1%
Low Positive 1	35**	1.2	0.1	4.4%	0.1	5.6%	0.0	1.4%	0.0	3.9%	0.1	8.3%
Low Positive 2	36	1.4	0.1	5.8%	0.1	4.8%	0.0	0.0%	0.0	2.4%	0.1	7.9%
Positive Control	36	2.0	0.1	5.5%	0.1	2.8%	0.1	3.0%	0.3	15.9%	0.3	17.2%

Table 12: Reproducibility Results; BioPlex 2200 EBV VCA IgM

*Between site variance includes between lot variance.

**1 replicate missing due to insufficient sample volume.

		Table 13: Reproducibility Results; BioPlex 2200 Heterophile
--	--	-------------------------------------------------------------	--

HeterophilePanel Members	SampleN	GrandMeanAl	Within-Run		Between-Day		Between-Run		Between-Site*		Total
			SD	%CV	SD	%CV	SD	%CV	SD	%CV	SD	%CV
High Positive 1	36	2.8	0.1	2.8%	0.0	0.0%	0.1	5.2%	0.1	5.1%	0.2	7.8%
High Positive 2	36	2.7	0.1	3.2%	0.0	0.0%	0.1	4.4%	0.0	0.7%	0.1	5.5%
Low Positive 1	35**	1.9	0.1	5.0%	0.0	0.0%	0.0	0.0%	0.0	2.3%	0.1	5.5%
Low Positive 2	36	1.8	0.1	4.7%	0.0	1.0%	0.0	0.0%	0.1	5.6%	0.1	7.4%
Positive Control	36	2.5	0.1	5.3%	0.1	2.6%	0.0	0.0%	0.0	0.0%	0.1	5.9%

*Between site variance includes between lot variance.

** 1 replicate missing due to insufficient sample volume.

C. Precision Studies

A precision panel, consisting of six (6) panel members was prepared by Bio-Rad Laboratories. Two (2) of the six (6) panel members had high levels of the antibodies contained in the BioPlex 2200 EBV IgG kit EBV IgM kit (EBV VCA IgM and Heterophile) and two (2) of the six (6) panel members had antibody levels near the cutoff, both prepared from positive patient samples. Two (2) of the six (6) panel members were negative (one high negative and one low negative) for both of the analytes.

Precision testing was performed at Bio-Rad Laboratories on one lot of the EBV IgM kit, one lot of the EBV IgM Calibrator Set and one lot of the EBV IgM Control Set. Each of the six (6) panel members was tested in duplicate (x2) on two (2) runs per day for ten (10) days using one (1) lot of EBV IgM kit, one (1) lot of EBV IgM Calibrator Set and one (1) lot of EBV IgM Control Set (2 times x 2 runs x 10 days = 40 replicates per panel member). The data were analyzed for intraassay and inter-assay precision according to the principles described in the Clinical Laboratory Standards Institute guidance EP5-A2, revised November 2004 and ISO/TR 22971:2205. The standard deviation (SD) and percent coefficient of variation (%CV) were calculated.

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Image /page/10/Picture/0 description: The image shows the logo for Bio-Rad. The logo is white text on a black background. The text is in all capital letters and is bolded.

EBV VCA IgMPanel Members	SampleN*	AIMean	Within-Run		Between-Day		Between-Run		Total
			SD	%CV	SD	%CV	SD	%CV	SD	%CV
High Positive 1	42	2.5	0.2	8.8%	0.1	2.4%	0.2	6.6%	0.3	11.2%
High Positive 2	43	2.7	0.2	5.6%	0.2	6.9%	0.1	1.8%	0.2	9.1%
Low Positive 1	42	1.6	0.1	6.5%	0.0	0.0%	0.1	4.3%	0.1	7.8%
Low Positive 2	42	1.9	0.1	4.4%	0.1	2.9%	0.1	4.8%	0.1	7.1%
High Negative	43	0.7	0.0	6.4%	0.0	0.0%	0.0	2.7%	0.0	7.0%
Low Negative	43	0.1	0.0	0.0%	0.0	0.0%	0.0	0.0%	0.0	0.0%

Table 14: Precision Results; BioPlex 2200 EBV VCA IgM

Table 15: Precision Results; BioPlex 2200 Heterophile

HeterophilePanel Members	SampleN*	AIMean	Within-Run		Between-Day		Between-Run		Total
			SD	%CV	SD	%CV	SD	%CV	SD	%CV
High Positive 1	42	2.8	0.1	3.7%	0.2	8.6%	0.1	1.8%	0.3	9.5%
High Positive 2	43	2.8	0.1	4.7%	0.3	9.9%	0.1	5.3%	0.3	12.2%
Low Positive 1	42	2.1	0.1	3.9%	0.1	3.7%	0.2	9.0%	0.2	10.5%
Low Positive 2	42	1.9	0.1	3.9%	0.1	4.6%	0.1	7.5%	0.2	9.6%
High Negative	43	0.7	0.0	7.0%	0.0	4.6%	0.0	0.0%	0.1	8.4%
Low Negative	43	0.0	0.0	0.0%	0.0	0.0%	0.0	0.0%	0.0	0.0%

D. Comparative Testing

Typical Antibody Response Characterization

The following table demonstrates a generally accepted algorithm for classifying patients into an EBV status via serologic profiles. EBV status can be applied to any patient based on results of standard tests. In acute IM, both EBV IgM and EBV IgG antibodies to viral capsid antigen (VCA) rise rapidly. EBV VCA IgM antibody disappears over about four weeks. Heterophile antibody, which is of the IgM class, appears only during acute infection and fades rapidly over about four weeks. EBV EA-D IgG antibody shows a transient raise during acute infection, undetectable after 3 - 6 months. EBV NA-1 IgG antibody usually appears 3 months after initial infection and remains for life.

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Image /page/11/Picture/0 description: The image shows the Bio-Rad logo. The logo is white text on a black background. The background is a rounded rectangle.

Table 16: Serological Status

EBV Serological Status	EBV NA-1 IgG	EBV VCA IgG	EBV EA-D IgG	EBV VCA IgM	Heterophile Antibody
	Neg (-)	Pos (+)	Pos (+)	Pos (+)	Neg (-)
	Neg (-)	Neg (-)	Pos (+)	Pos (+)	Pos (+)
	Neg (-)	Pos (+)	Neg (-)	Pos (+)	Pos (+)
Primary Acute	Neg (-)	Neg (-)	Neg (-)	Pos (+)	Pos (+)
	Neg (-)	Neg (-)	Neg (-)	Pos (+)	Neg (-)
	Neg (-)	Neg (-)	Pos (+)	Pos (+)	Neg (-)
	Neg (-)	Pos (+)	Pos (+)	Pos (+)	Pos (+)
	Neg (-)	Pos (+)	Pos (+)	Neg (-)	Pos (+)
	Neg (-)	Pos (+)	Neg (-)	Pos (+)	Neg (-)
	Pos (+)	Pos (+)	Pos (+)	Pos (+)	Pos (+)
	Pos (+)	Pos (+)	Pos (+)	Pos (+)	Neg (-)
Late Acute	Pos (+)	Pos (+)	Neg (-)	Pos (+)	Pos (+)
	Pos (+)	Pos (+)	Pos (+)	Neg (-)	Neg (-)
	Pos (+)	Pos (+)	Neg (-)	Pos (+)	Neg (-)
Recovering	Neg (-)	Pos (+)	Pos (+)	Neg (-)	Neg (-)
	Neg (-)	Pos (+)	Neg (-)	Neg (-)	Neg (-)
Previous Infection	Pos (+)	Pos (+)	Neg (-)	Neg (-)	Neg (-)
Susceptible	Neg (-)	Neg (-)	Neg (-)	Neg (-)	Neg (-)

Notes: For the purposes of serological characterization, equivocal results were considered negative. Any serological pattern not identified in Table 16 should be considered inconclusive.

Comparison of BioPlex 2200 EBV IgM kit and Microplate EIA/Agglutination Tests Performance of the EBV IgM kit was tested against corresponding commercially available microplate EIA/agglutination tests. A total of 621 banked serum samples from patients for which an EBV test was ordered were tested at 3 U.S. clinical testing sites. The EBV IgG kit was run in conjunction with the EBV IgM kit to allow for a complete antibody response profile. The characterization by antibody response was not compared with clinical data regarding presence, absence or status of disease. Two (2) samples were excluded due to RBB analysis error messages during BioPlex 2200 EBV IgM testing, One (1) sample was excluded due to RBB analysis error messages during BioPlex 2200 EBV IgG testing. Using Table 16 as a guideline, results were analyzed by BioPlex 2200 EBV IgM analytes and corresponding EBV IgM reference assays according to serological characterization based on reference assay results. For the purpose of percent agreement calculations, BioPlex 2200 EBV IgM equivocal results were assigned to the opposite clinical interpretation than that of the corresponding reference assay result. Results from all sites are shown and summarized in Tables 17 - 20.

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Image /page/12/Picture/0 description: The image shows the logo for Bio-Rad Laboratories. The logo is white text on a black, rounded rectangle. The text is in a bold, sans-serif font.

EBVSerological Status	Reference EBV VCA IgM InterpretationPositiveBioPlex 2200 EBV VCA IgM			NegativeBioPlex 2200 EBV VCA IgM			Total
	PosN	EqvN	NegN	PosN	EqvN	NegN
Primary Acute	30	1	0	0	0	0	31
Late Acute	31	1	16	1	4	57	110
Recovering	0	0	0	1	0	3	4
Previous Infection	0	0	0	6	6	293	305
Susceptible	0	0	0	4	0	123	127
Inconclusive	4	0	0	6	0	31	41
Overall	65	2	16	18	10	507	618

Table 17. BioPlex 2200 EBV VCA IgM vs. EIA: Comparison by Serological Pattern Characterization

Table 18: BioPlex 2200 EBV VCA IgM vs. EIA: Percent Agreement & Confidence Intervals by Serological Pattern Characterization

EBVSerological Status	Positive Agreement	95% CI	Negative Agreement	95% CI
Primary Acute	(30/31)	96.8%	83.8 - 99.4%	(0/0)	N/A*	N/A*
Late Acute	(31/48)	64.6%	50.4 - 76.6%	(57/62)	91.9%	82.5 - 96.5%
Recovering	(0/0)	N/A*	N/A*	(3/4)	75.0%	30.1 - 95.4%
Previous Infection	(0/0)	N/A*	N/A*	(293/305)	96.1%	93.2 - 97.7%
Susceptible	(0/0)	N/A*	N/A*	(123/127)	96.9%	92.2 - 98.8%
Inconclusive	(4/4)	100%	51.0-100%	(31/37)	83.8%	68.9 - 92.3%
Overall	(65/83)	78.3%	68.3 - 85.8%	(507/535)	94.8%	92.5 - 96.4%

*In cases where agreement resulted in (0/0) samples, percent and 95% confidence interval could not be calculated.

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Image /page/13/Picture/0 description: The image shows the logo for Bio-Rad. The logo is white text on a black rounded rectangle. The text is in all caps and reads "BIO-RAD".

Table 19: BioPlex 2200 Heterophile vs. Agglutination Test: Comparison by Serological Pattern Characterization

	Reference Heterophile Interpretation
EBVSerological Status	Positive			Negative			Total
	BioPlex 2200 Heterophile			BioPlex 2200 Heterophile
	Pos	Eqv	Neg	Pos	Eqv	Neg
	N	N	N	N	N	N	N
Primary Acute	16	1	2	2	0	10	31
Late Acute	3	0	1	1	1	104	110
Recovering	0	0	0	0	0	4	4
Previous Infection	0	0	0	0	3	302	305
Susceptible	0	0	0	0	0	127	127
Inconclusive	4	0	23	0	0	14	41
Overall	23	1	26	3	4	561	618

Table 20: BioPlex 2200 Heterophile vs. Agglutination Test: Percent & Confidence Intervals by Serological Pattern Characterization

EBVSerological Status	Positive Agreement	95% CI	Negative Agreement	95% CI
Primary Acute	(16/19)	62.4 - 94.5%	(10/12)	55.2 - 95.3%
Late Acute	(3/4)	30.1 - 95.4%	(104/106)	93.4 - 99.5%
Recovering	(0/0)	N/A*	(4/4)	51.0 - 100%
Previous Infection	(0/0)	N/A*	(302/305)	97.1 - 99.7%
Susceptible	(0/0)	N/A*	(127/127)	97.1 - 100%
Inconclusive	(4/27)	5.9 - 32.5%	(14/14)	78.5 - 100%
Overall	(23/50)	33.0 - 59.6%	(561/568)	97.5 - 99.4%
	84.2%		83.3%
	75.0%		98.1%
	N/A*		100%
	N/A*		99.0%
	N/A*		100%
	14.8%		100%
	46.0%		98.8%

*In cases where agreement resulted in (0/0) samples, percent and 95% confidence interval could not be calculated.

Comparison of BioPlex 2200 EBV IgM kit and Microplate EIA (known VCA IgM positive samples) Performance of the EBV IgM kit was further tested with serum samples that were previously characterized as EBV VCA IgM positive samples by another commercially available IgM EIA. A total of 100 purchased EBV VCA IgM positive samples were tested with the EBV IgM kit against corresponding commercially available microplate EIA/agglutination tests at a U.S. clinical testing site. The EBV IgG kit was run in conjunction with the EBV IgM kit to allow for a complete antibody response profile. The characterization by antibody response was not compared with clinical data regarding presence, absence or status of disease. Using Table 16 as a guideline, results were analyzed by BioPlex 2200 EBV IgM analytes and corresponding EBV IgM reference assays according to serological characterization based on reference assay results. For the purpose of percent agreement calculations, BioPlex 2200 EBV IgM equivocal results were assigned to the opposite clinical interpretation than that of the corresponding reference assay result. Results are shown and summarized in Tables 21 - 25.

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Image /page/14/Picture/0 description: The image shows the logo for Bio-Rad. The logo is white text on a black background. The text is in a bold, sans-serif font and is slightly stylized.

EBV SerologicalStatus	Reference EBV VCA IgM InterpretationPositiveBioPlex 2200 EBV VCA IgM			Reference EBV VCA IgM InterpretationNegativeBioPlex 2200 EBV VCA IgM			Total
	Pos	Eqv	Neg	Pos	Eqv	Neg
	N	N	N	N	N	N	N
Late acute	100	0	0	0	0	0	100
Overall	100	0	0	0	0	0	100

Table 21: BioPlex 2200 EBV VCA IgM vs. EIA: Comparison by Serological Pattern Characterization

Table 22: BioPlex 2200 EBV VCA IgM vs. EIA: Percent Agreement & Confidence Intervals by Serological Pattern Characterization

EBV SerologicalStatus	PositiveAgreement	95% CI	NegativeAgreement	95% CI
Late acute	(100/100)	100%	96.3 - 100%	(0/0)	NA*
Overall	(100/100)	100%	96.3 - 100%	(0/0)	NA*

*In cases where agreement resulted in (0/0) samples, percent and 95% confidence interval could not be calculated.

Table 23: BioPlex 2200 Heterophile vs. Agglutination Test: Comparison by Serological Pattern Characterization

EBV SerologicalStatus	Reference Heterophile Interpretation						Total
	Positive			Negative
	BioPlex 2200 Heterophile	BioPlex 2200 Heterophile
	Pos	Eqv	Neg	Pos	Eqv	Neg
	N	N	N	N	N	N	N
Late acute	100	0	0	0	0	0	100
Overall	100	0	0	0	0	0	100

Table 24: BioPlex 2200 Heterophile vs. Agglutination Test: Percent & Confidence Intervals by Serological Pattern Characterization

EBV SerologicalStatus	PositiveAgreement	95% CI	NegativeAgreement	95% CI
Late acute	(100/100)	100%	96.3 - 100%	(0/0)	NA*
Overall	(100/100)	100%	96.3 - 100%	(0/0)	NA*

*In cases where agreement resulted in (0/0) samples, percent and 95% confidence interval could not be calculated.

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Image /page/15/Picture/0 description: The image shows the logo for BIO-RAD. The logo is white text on a black rounded rectangle. The text is in all caps and bolded.

Comparison of EBV Serological Status

Using Table 16 as a quideline, samples characterized into serological status associated with EBV disease, using the commercially available microplate EIA and agglutination tests, were compared with characterizations using BioPlex 2200 EBV IgG and IgM kits. The BioPlex 2200 EBV IgM kit was run in conjunction with the BioPlex 2200 EBV IgG kit to allow for a complete antibody response profile. The characterization by antibody response was not compared with clinical data regarding presence, absence or status of disease. Results from 618 serum samples tested at 3 U.S. clinical testing sites are shown in Table 25.

EBV Serological status		BioPlex 2200 EBV IgG & IgM Profile							% SerologicalAgreement	95%ConfidenceInterval
		Primary Acute	Late Acute	Recovering	Previous Infection	Susceptible	Inconclusive	Total
CommerciallyAvailable Assays	Primary Acute	30	0	0	0	0	1	31	96.8%	83.8 - 99.4%
	Late Acute	5	90	1	13	0	1	110	81.8%	73.6 - 87.9%
	Recovering	1	0	3	0	0	0	4	75.0%	30.0 - 95.4%
	Previous Infection	0	31	2	263	4	5	305	86.2%	81.9 - 89.7%
	Susceptible	4	0	0	0	122	1	127	96.1%	91.1 - 98.3%
	Inconclusive	6	10	0	7	11	7	41	17.1%	8.5 - 31.3%
	Overall	46	131	6	283	137	15	618	83.3%	80.2 - 86.1%

Table 25: Comparison of EBV Serological Status

Note: Calculations are performed for unshaded areas only.

Comparison of Acute and Non-acute EBV Serological Status

The results obtained from the summarized information provided in Table 26 were further classified into Acute Infection. Acute Infection includes Primary Acute and Late Acute. Non-Acute Infection includes Susceptible, Recovering and Previous Infection as defined in Table 16. Inconclusive includes any samples reactivity are not consistent with any category listed in Table 16. Results are summarized in Table 26.

Table 26: Acute vs. Non-acute
-------------------------------

EBV Serological status	Acute	Non-Acute	Inconclusive	Total	BioPlex 2200 EBV IgG & IgM Profile% SerologicalAgreement	95% ConfidenceInterval
Acute	125	14	2	141	88.7%	82.4 - 92.9%
Non-Acute	36	394	6	436	90.4%	87.2 - 92.8%
Inconclusive	16	18	7	41	17.1%	8.5 - 31.3%
Overall	177	426	15	618	85.1%	82.1 - 87.7%

Note: Calculations are performed for unshaded areas only.

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Image /page/16/Picture/0 description: The image shows the logo for Bio-Rad. The text "BIO-RAD" is in white, bold, sans-serif font. The text is set against a black, rounded rectangle.

Comparison of EBV Serological Status (known IgM positive samples)

Using Table 16 as a quideline, samples characterized into serological status associated with EBV disease, using the commercially available microplate EIA and agglutination tests, were compared with characterizations using BioPlex 2200 EBV IgG and IgM kits. The BioPlex 2200 EBV IgM kit was run in conjunction with the BioPlex 2200 EBV IgG kit to allow for a complete antibody response profile. The characterization by antibody response was not compared with clinical data regarding presence, absence or status of disease. A total of 100 purchased EBV VCA IgM positive samples were tested at a U.S. clinical testing site. Results are shown in Table 27.

EBV Serological status		Late Acute	Total	% Serological Agreement	95% Confidence Interval
Commercially Available Assays	Primary Acute	0	0	N/A	N/A
	Late Acute	100	100	100%	96.3-100%
	Recovering	0	0	N/A	N/A
	Previous Infection	0	0	N/A	N/A
	Susceptible	0	0	N/A	N/A
	Inconclusive	0	0	N/A	N/A
	Overall	100	100	100%	96.3-100%

Table 27: Comparison of EBV Serological Status (known VCA IgM positive samples)

Note: Calculations are performed for unshaded areas only.

Comparison of Acute and Non-acute EBV Serological Status (known VCA IgM positive samples) The results obtained from the summarized information provided in Table 27 were further classified into two groups; Acute Infection and Non-Acute Infection. Acute Infection includes Primary Acute and Late Acute. Non-Acute Infection includes samples characterized as Susceptible, Recovering and Previous Infection as defined in Table 16. Inconclusive includes any samples whose patterns of antibody reactivity are not consistent with any category listed in Table 16. Results are summarized in Table 28.

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Image /page/17/Picture/0 description: The image shows the logo for BIO-RAD. The logo is white text on a black rounded rectangle. The text is in a bold, sans-serif font.

	EBV Serological status	Acute	Total	% Serological Agreement	95% Confidence Interval
CommerciallyAvailable Assays	Acute	100	100	100%	96.3-100%
	Non-Acute	0	0	N/A	N/A
	Inconclusive	0	0	N/A	N/A
	Overall	100	100	100%	96.3-100%

Table 28: Acute vs. Non-acute (known VCA IgM positive samples)

Note: Calculations are performed for unshaded areas only.

Primary Acute Serological Status by Age

The results from Table 28 with a Primary Acute serological status were further analyzed by age group. Results are summarized in Table 29.

Table 29: Primary Acute Serological Status by Age

EBV Primary AcuteInfection Grouped byAge	N	BioPlex 2200 EBV VCA IgM			BioPlex 2200 Heterophile
		Pos (+)	(%)	95% CI	Pos (+)	(%)	95% CI
< 5 Yrs	4	4	100%	51.0 - 100 %	0	0%	N/A
5 - 12 Yrs	7	7	100%	64.6 - 100 %	5*	71%	35.9 - 91.8%
13 - 20 Yrs	14	13	93%	68.5 - 98.7%	10	71%	45.4 - 88.3%
Adult ≥ 21 Yrs	6	6	100%	61.0 - 100%	3**	50%	18.8 - 81.2%
Total	31	30	97%	83.8 - 99.4%	18	58%	40.8 - 73.6%

*Two (2) samples were BioPlex 2200 positive, commercially available Heterophile Agglutination negative. **One (1) sample was commercially available Heterophile Agglutination positive, BioPlex 2200 negative.

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D. Cross-Reactivity

A cross-reactivity study was performed to determine if samples from various disease states and other potentially interfering factors interfere with test results when tested with the BioPlex 2200 EBV IqM kit. A panel of at least ten (10) specimens* positive for each cross reactant were evaluated for possible cross reactivity with the BioPlex 2200 EBV IgM kit for both EBV IgM antibody assays. The test specimens were also evaluated on corresponding commercially available microplate EIA and latex agglutination tests. Due to the number of VCA IgM positive results obtained with the initial 10 Toxoplasmosis and CMV samples, an additional 33 toxoplasmosis and 42 CMV positive samples were tested with the BioPlex 2200 EBV IqM kit. This study was performed to demonstrate that the BioPlex 2200 VCA 1gM assay does not exhibit cross reactivity with Toxoplasmosis or CMV IdM samples. For these additional samples, only those that exhibited positive reactivity were tested on corresponding commercially available microplate EIA and latex agglutination tests. Most of the samples evaluated were high positive for each disease state. The majority of all samples that elicited a positive result were also confirmed positive by the corresponding commercially available test, indicating reactivity to EBV IgM antibodies rather than cross reactivity with a potentially interfering factor. Results can be found in Table 30.

*Due to limited availability of samples, only four E. coli specimens were evaluated.

CrossReactives	N	Method	BioPlex 2200EBV IgM		CrossReactives	N	Method	BioPlex 2200EBV IgM
			EBV VCA	Heterophile				EBV VCA	Heterophile
ANA	10	BioPlex 2200	1	0	RheumatoidFactor	10	BioPlex 2200	0	0
		Commercial Assay	1	0			Commercial Assay	0	0
		Discrepants	0	0			Discrepants	0	0
Rubella IgM	10	BioPlex 2200	0	0	VZV IgM	10	BioPlex 2200	0	0
		Commercial Assay	0	0			Commercial Assay	0	0
		Discrepants	0	0			Discrepants	0	0
HSV IgM	10	BioPlex 2200	0	0	HIV	10*	BioPlex 2200	0	0
		Commercial Assay	0	0			Commercial Assay	N/A	N/A
		Discrepants	0	0			Discrepants	N/A	N/A
E. Coli	4*	BioPlex 2200	0	0	Pregnantwomen	10	BioPlex 2200	0	0
		Commercial Assay	N/A	N/A			Commercial Assay	0	0
		Discrepants	N/A	N/A			Discrepants	0	0
Toxo IgM		BioPlex 2200 (N)	43	43	CMV IgM		BioPlex 2200 (N)	52	52
		BioPlex 2200 (+)	14	0			BioPlex 2200 (+)	25	1
		Commercial Assay (N)	14†	10††			Commercial Assay (N)	25†	10††
		Commercial Assay (+)	9	0			Commercial Assay (+)	22	0
		Discrepants	5	0			Discrepants	3	1

Table 30: Cross-Reactivity

*Commercially available assay data was not obtained, due to low sample volume.

+Commercially available assay testing was performed on BioPlex 2200 positive samples only. tt Commercially available assay testing was performed on the initial ten samples evaluated.

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Image /page/19/Picture/1 description: The image shows the logo for the U.S. Department of Health & Human Services. The logo features a stylized eagle with three tail feathers. The eagle is facing to the right. The text "DEPARTMENT OF HEALTH & HUMAN SERVICES - USA" is arranged in a circle around the eagle.

Food and Drug Administration 2098 Gaither Road Rockville MD 20850

Mr. David Bhend Regulatory Affairs Associate Bio-Rad Laboratories, Inc. Diagnostics Group 6565 18510 Ave, N.E. Redmond, WA 98052

DEC - 8 2006

K062213 Re:

Trade/Device Name: BioPlex 2200 EBV IgM Panel on the BioPlex 2200 Multi-Analyte Detection System BioPlex 2200 EBV IgM Control Set BioPlex 2200 EBV IgM Calibrator Set Regulation Number: 21 CFR 866.5640 Regulation Name: Infectious mononucleosis immunological test system. Regulatory Class: Class II Product Code: LSE, KTN Dated: November 13, 2006 Received: November 14, 2006

Dear Mr. Bhend:

We have reviewed your Section 510(k) premarket notification of intent to market the device referenced above and have determined the device is substantially equivalent (for the indications for use stated in the enclosure) to legally marketed predicate devices marketed in interstate commerce prior to May 28, 1976, the enactment date of the Medical Device Amendments, or to devices that have been reclassified in accordance with the provisions of the Federal Food, Drug, and Cosmetic Act (Act) that do not require approval of a premarket approval application (PMA). You may, therefore, market the device, subject to the general controls provisions of the Act. The general controls provisions of the Act include requirements for annual registration, listing of devices, good manufacturing practice, labeling, and prohibitions against misbranding and adulteration.

If your device is classified (see above) into either class II (Special Controls) or class III (PMA), it may be subject to such additional controls. Existing major regulations affecting your device can be found in Title 21, Code of Federal Regulations (CFR), Parts 800 to 895. In addition, FDA may publish further announcements concerning your device in the Federal Register.

Please be advised that FDA's issuance of a substantial equivalence determination does not mean that FDA has made a determination that your device complies with other requirements of the Act or any Federal statutes and regulations administered by other Federal agencies. You must comply with all the Act's requirements, including, but not limited to: registration and listing (21 CFR Part 807); labeling (21 CFR Parts 801 and 809); and good manufacturing practice requirements as set forth in the quality systems (QS) regulation (21 CFR Part 820).

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This letter will allow you to begin marketing your device as described in your Section 510(k) premarket notification. The FDA finding of substantial equivalence of your device to a legally marketed predicate device results in a classification for your device and thus, permits your device to proceed to the market.

If you desire specific information about the application of labeling requirements to your device, or questions on the promotion and advertising of your device, please contact the Office of In Vitro Diagnostic Device Evaluation and Safety at (240) 276-0484. Also, please note the regulation entitled, "Misbranding by reference to premarket notification" (21CFR Part 807.97). You may obtain other general information on your responsibilities under the Act from the Division of Small Manufacturers, International and Consumer Assistance at its toll-free number (800) 638-2041 or (240) 276-3150, or at its Internet address http://www.fda.gov/cdrh/dsma/dsmamain.html.

Sincerely yours,

Sala Hans

Sally A. Hojvat, M.Sc., Ph.D. Director Division of Microbiology Devices Office of In Vitro Diagnostic Device Evaluation and Safety Center for Devices and Radiological Health

Enclosure

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INDICATIONS FOR USE STATEMENT

510(k) Number: K062213

BioPlex 2200 EBV IqM Kit on the BioPlex 2200 Multi-Analyte Device Name: Detection System BioPlex 2200 EBV IgM Control Set BioPlex 2200 EBV IgM Calibrator Set

Indications for Use:

BioPlex 2200 EBV IqM Kit

The EBV IgM kit is intended for use with the Bio-Rad BioPlex 2200 System.

BioPlex 2200 EBV IgM Calibrator Set

The BioPlex 2200 EBV IgM Calibrator Set is intended for the calibration of the BioPlex 2200 EBV IgM Reagent Pack.

BioPlex 2200 EBV IgM Control Set

X ----------------------------------------------------------------------------------------------------------------------------------------------------------------------------Prescription Use: (Per 21 CFR 801.109)

AND/OR

Over-The-Counter Use: (Optional Format 1-2-96)

(PLEASE DO NOT WRITE BELOW THIS LINE -- CONTINUE ON ANOTHER PAGE IF NEEDED)

Office of Device Evaluation (ODE) Concur Division Siein-Off

Office of In Vitro Diagnostic Device Evaluation and Safety

510/k) 1206221

Regulation Number and Section

§ 866.3235 Epstein-Barr virus serological reagents.

(a)
Identification. Epstein-Barr virus serological reagents are devices that consist of antigens and antisera used in serological tests to identify antibodies to Epstein-Barr virus in serum. The identification aids in the diagnosis of Epstein-Barr virus infections and provides epidemiological information on diseases caused by these viruses. Epstein-Barr viruses are thought to cause infectious mononucleosis and have been associated with Burkitt's lymphoma (a tumor of the jaw in African children and young adults) and postnasal carcinoma (cancer).(b)
Classification. Class I (general controls).