DIASORIN ETI MAK-2 PLUS ASSAY

P990038 · DiaSorin, Inc. · LOM · Mar 30, 2001 · Microbiology

Device Facts

• Record ID: P990038
• Device Name: DIASORIN ETI MAK-2 PLUS ASSAY
• Applicant: DiaSorin, Inc.
• Product Code: LOM · Microbiology
• Decision Date: Mar 30, 2001
• Decision: APWD
• Regulation: 21 CFR 866.3172
• Device Class: Class 2

Indications for Use

ETI-MAK-2 PLUS is an in vitro enzyme immunoassay (EIA) intended for use in the qualitative determination of hepatitis B surface antigen (HBsAg) in human serum or plasma (EDTA, citrate or heparin). The ETI-MAK-2 PLUS is intended for manual use and with the Biochem Immunosystems Labotech/ETI-LAB automated instrument. The detection of HBsAg is indicative of a laboratory diagnosis for hepatitis B virus (HBV) infection, either acute or chronic. Further HBV serological marker testing is required to define the specific disease state. The ETI-MAK-2 PLUS assay’s performance has not been established for the monitoring of HBV disease or therapy.

Device Story

ETI-MAK-2 PLUS is an enzyme immunoassay (EIA) for qualitative HBsAg detection in human serum or plasma. The device uses polystyrene microwells coated with mouse monoclonal antibodies to capture HBsAg. Captured antigen is detected via a horseradish peroxidase-labeled sheep antibody tracer. Addition of TMB chromogen/substrate produces a blue color (650 nm) proportional to HBsAg concentration, turning yellow (450 nm) after stop solution. Absorbance is measured spectrophotometrically; results are compared to a cutoff derived from calibrators. The assay is performed manually or using the Biochem Immunosystems Labotech/ETI-LAB automated instrument. Results aid clinicians in diagnosing acute or chronic HBV infection. The device provides diagnostic information for patients suspected of HBV infection, including pregnant women and high-risk populations, facilitating clinical management decisions.

Clinical Evidence

Clinical performance evaluated via prospective (n=136) and retrospective (n=650) studies, plus pregnant women cohort (n=564). Prospective samples showed 100% positive agreement for chronic infection and 98.9% negative agreement for non-infected individuals. Retrospective studies demonstrated high agreement across various HBV classifications (acute, chronic, recovery). Analytical sensitivity is 0.1 PEI U/mL. Bench testing confirmed reproducibility, cross-reactivity (no interference from common viral/bacterial proteins), and stability (6 months). No clinical data for Labotech instrument reproducibility provided at approval; post-approval study required.

Technological Characteristics

Direct non-competitive enzyme immunoassay (EIA). Components: polystyrene microwells coated with mouse monoclonal anti-HBs (IgG1-k), HRP-labeled sheep anti-HBs tracer, TMB/H2O2 chromogen/substrate, 1N sulfuric acid stop solution. Manual or automated (Labotech/ETI-LAB) processing. Connectivity: standalone or integrated with automated microplate processor. Preservative: 0.2% ProClin 300.

Indications for Use

Indicated for qualitative detection of HBsAg in human serum or plasma to aid in the laboratory diagnosis of acute or chronic hepatitis B virus (HBV) infection. Intended for use in clinical laboratory settings.

Regulatory Classification

Identification

A qualitative hepatitis B virus (HBV) antigen assay is identified as an in vitro diagnostic device intended for prescription use for qualitative use with human serum, plasma, or other matrices that aids in the diagnosis of chronic or acute HBV infection. HBV surface antigen (HbsAg) is also used for screening of HBV infection in pregnant women to identify neonates who are at risk of acquiring hepatitis B during perinatal period. The assay is not intended for screening of blood, plasma, cells, or tissue donors.

Special Controls

*Classification.* Class II (special controls). The special controls for this device are:(1) The labeling required under § 809.10(b) of this chapter must include: (i) A prominent statement that the assay is not intended for the screening of blood, plasma, cells, or tissue donors. (ii) A detailed explanation of the principles of operation and procedures for performing the assay. (iii) A detailed explanation of the interpretation of results. (iv) Limitations, which must be updated to reflect current clinical practice and disease presentation and management. The limitations must include statements that indicate: (A) The specimen types for which the device has been cleared, and that use of this assay with specimen types other than those specifically cleared for this device may result in inaccurate assay results. (B) When appropriate, performance characteristics of the assay have not been established in populations of immunocompromised or immunosuppressed patients or other populations where assay performance may be affected. (C) Diagnosis of hepatitis B infection should not be established on the basis of a single assay result but should be determined by a licensed healthcare professional in conjunction with the clinical presentation, history, and other diagnostic procedures. (D) Detection of HBV antigens indicates a current infection with hepatitis B virus but does not differentiate between acute or chronic infection. False reactive HbsAg result may occur for up to 2 weeks after vaccination with HbsAg containing vaccine. (E) Current methods for the detection of hepatitis B antigens may not detect all potentially infected individuals. A non-reactive assay result does not exclude the possibility of exposure to or infection with hepatitis B virus. A non-reactive assay result in individuals with prior exposure to hepatitis B may be due to but not limited to antigen levels below the detection limit of this assay or lack of antigen reactivity to the antibodies in this assay. HBV mutants lacking the ability to produce antigens have been reported. These may occur as “escape” mutants in the presence of anti-HBV antibodies and such patients may be infectious. (F) Results obtained with this assay may not be used interchangeably with results obtained with a different manufacturer's assay. (2) Design verification and validation must include the following: (i) A detailed device description, including all parts that make up the device, ancillary reagents required but not provided, an explanation of the device methodology, design of the capture antibody(ies), external controls, and computational path from collected raw data to reported result ( *e.g.,* how collected raw signals are converted into a reported signal and result), as applicable to the detection method and device design.(ii) For devices with assay calibrators, the design and composition of all primary, secondary, and subsequent quantitation standards used for calibration as well as their traceability to a standardized reference material that FDA has determined is appropriate ( *e.g.,* a recognized consensus standard). In addition, analytical testing must be performed following the release of a new lot of the standard material that was used for device clearance or approval, or when there is a transition to a new calibration standard.(iii) Documentation and characterization ( *e.g.,* supplier, determination of identity, purity, and stability) of all critical reagents (including description of the capture antibody(ies)), and protocols for maintaining product integrity throughout its labeled shelf life.(iv) Risk analysis and management strategies, such as Failure Modes Effects Analysis and/or Hazard Analysis and Critical Control Points summaries and their impact on assay performance. (v) Final release criteria to be used for manufactured assay lots with appropriate evidence that lots released at the extremes of the specifications will meet the identified analytical and clinical performance characteristics as well as stability. (vi) Stability studies for reagents must include documentation of an assessment of real-time stability for multiple reagent lots using the indicated specimen types and must use acceptance criteria that ensure that analytical and clinical performance characteristics are met when stability is assigned based on the extremes of the acceptance range. (vii) All stability protocols, including acceptance criteria. (viii) Final release assay results for each lot used in clinical studies. (ix) Reproducibility study data that includes the testing of three independent production lots. (x) Detailed documentation of analytical performance studies conducted, as appropriate to the technology, specimen types tested, and intended use of the device, including, the limit of blank (LoB), limit of detection (LoD), cutoff, precision (reproducibility) including lot-to-lot and/or instrument-to-instrument precision, interference, cross reactivity, carryover, hook effect, seroconversion panel testing, matrix equivalency, prominent mutants/variants detection ( *e.g.,* for HbsAg), specimen stability, reagent stability, and cross-genotype antigen detection sensitivity, when appropriate.(xi) Analytical sensitivity of the assay that is the same or better than that of other cleared or approved assays. (xii) For devices with associated software or instrumentation, documentation must include a detailed description of device software, including software applications and hardware-based devices that incorporate software. The detailed description must include documentation of verification, validation, and hazard analysis and risk assessment activities, including an assessment of the impact of threats and vulnerabilities on device functionality and end users/patients as part of cybersecurity review. (xiii) Detailed documentation and results from a clinical study. Performance must be analyzed relative to an FDA cleared or approved HBV antigen assay or a comparator that FDA has determined is appropriate. This study must be conducted using appropriate patient samples, with an appropriate number of HBV reactive and non-reactive samples in applicable risk and disease categories, and any applicable confirmatory testing. Additional relevant patient groups must be validated as appropriate. The samples must include prospective (sequential) samples for each identified specimen type and, as appropriate, additional characterized clinical samples. Samples must be sourced from geographically diverse areas. This study must be conducted in the appropriate settings by the intended users to demonstrate clinical performance.

Submission Summary (Full Text)

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# SUMMARY OF SAFETY AND EFFECTIVENESS DATA

## 1. General Information

1.1. Device Generic Name: Immunoassay for hepatitis B surface antigen

1.2. Device Trade Name: ETI-MAK-2 PLUS

1.3. Applicant’s Name and Address:

DiaSorin
Via Crescentino
Saluggia (VC) 13040, Italy

1.4. U.S. Representative:

Sienna Partners, LLC
P.O. Box 103
Baldwin, MD 21013

1.5. PMA Number: P990038

1.6. Date of Panel Recommendation: January 20, 2000

1.7. Date of Notice of Approval to the Applicant: March 30, 2001

## 2. Indications For Use

ETI-MAK-2 PLUS is an in vitro enzyme immunoassay (EIA) intended for use in the qualitative determination of hepatitis B surface antigen (HBsAg) in human serum or plasma (EDTA, citrate or heparin). The ETI-MAK-2 PLUS is intended for manual use and with the Biochem Immunosystems Labotech/ETI-LAB automated instrument.

The detection of HBsAg is indicative of a laboratory diagnosis for hepatitis B virus (HBV) infection, either acute or chronic. Further HBV serological marker testing is required to define the specific disease state. The ETI-MAK-2 PLUS assay’s performance has not been established for the monitoring of HBV disease or therapy.

## 3. Device Description

3.1. Principle of The Assay

ETI-MAK-2 PLUS uses monoclonal antibodies to hepatitis B surface antigen (HBsAg) as the basis for this enzyme immunoassay. The assay is a direct, non-competitive test based on the use of polystyrene microwells coated with mouse monoclonal antibodies to HBsAg (directed to the “a” determinant of HBsAg). An enzyme tracer containing horseradish

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peroxidase-labeled sheep antibodies to HBsAg detects any captured HBsAg from the patient's specimen.

In the assay procedure, patient specimens and controls are incubated with incubation buffer in anti-HBs-coated microwells. If HBsAg is present in a specimen or control, it binds to the antibodies. Excess sample is removed by a wash step, and the enzyme tracer is then added to the microwells and allowed to incubate. The enzyme tracer binds to any antigen-antibody complexes present in the microwells. Excess enzyme tracer is removed by a wash step, and a chromogen/substrate solution is added to the microwells and allowed to incubate. If a sample contains HBsAg, the bound enzyme (horseradish peroxidase) chemically reduces the substrate peroxide, which concurrently oxidizes the chromogen tetramethylbenzidine (TMB) to a blue color (650 nm). The blue color turns to yellow (450 nm) after addition of the stop solution. If a sample does not contain HBsAg, the microwell will be colorless after the chromogen/substrate solution is added and will remain colorless after the stop solution is added. Color intensity, which is measured spectrophotometrically, is indicative of the concentration of HBsAg. Absorbance value readings for patient specimens are compared to a cutoff value determined from the mean of the calibrators.

## 3.2. Kit Configuration and Components

For detection of HBsAg, the ETI-MAK-2 PLUS system is comprised of the following:

Coated Strips
Microwells coated with mouse monoclonal antibodies to HBsAg (IgG1-k class, directed to the "a" determinant of HBsAg).

Enzyme Tracer
Horseradish peroxidase-labeled sheep IgG antibodies to HBsAg, buffer, protein stabilizers.
Preservative: 0.2% ProClin 300.

Tracer Diluent
Buffer, human serum/plasma, protein stabilizers.
Preservative: 0.2% ProClin 300.

Calibrator (Human)
Human serum/plasma non-reactive for HBsAg.
Preservative: 0.2% ProClin 300.

Negative Control (Human)
Human serum/plasma non-reactive for HBsAg.
Preservative: 0.2% ProClin 300.

Positive Control (Human)
Human serum/plasma reactive for HBsAg (subtypes ad and ay), protein stabilizers.
Preservative: 0.2% ProClin 300.

Incubation Buffer
Buffer, protein stabilizers, an inert blue dye.
Preservative: 0.2% ProClin 300.

Wash Buffer (concentrate)
Buffer, detergents, preservatives.

Chromogen/Substrate

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Tetramethylbenzidine/hydrogen peroxide system.
Stop Solution
1N sulfuric acid
Strip Sealers
Plate Sealers
Pouch Sealer

## 4. Contraindications

None

## 5. Warnings and Precautions

For in vitro diagnostic use only.

Warnings and precautions for users of the ETI-MAK-2 PLUS assay are stated in the product labeling.

## 6. Alternative Practices and Procedures

Historically, the presence of HBsAg, the main HBV serologic marker was determined using the immunodiffusion method. Since the method was very specific but with low sensitivity, other techniques such as counterimmunoelectrophoresis and complement fixation were developed. The advent of the newer technologies of complement fixation, counter immunoelectrophoresis, reversed passive hemagglutination, inhibition of passive hemagglutination for subtyping, RIA and enzyme-linked immunosorbent assay (ELISA) has yielded a further improvement of assay sensitivity. Currently, the commercially available kits are based on RIA or ELISA techniques that are similar in sensitivity and specificity.

## 7. Marketing History

The ETI-MAK-2 PLUS assay has never been marketed in the US or outside the US.

## 8. Potential Adverse Effects of the Device on Health

Failure of the product to perform as indicated or human error in use of the product may lead to a false result. A false negative result may be considered a patient or public health concern because the patient may be considered in early recovery and proceeding to HBV clearance. In this case, the risk may be that the patient's treatment may be altered or that they might not be considered infectious. This could lead to possible infection of uninfected individuals. A false positive result cannot be considered a patient or public health concern because the disease-state interpretation would be active HBV infection. In a false positive test, if HBsAg is the only hepatitis B serological marker detected, other additional testing should be done to validate the results e.g., testing new patient specimens or testing with an HBsAg confirmatory assay. This additional testing would invalidate the initial false positive test.

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# 9. Summary of Preclinical Studies

# 9.1. Comparison of Labotech Instrumentation with the Manual Assay

An instrument application study was conducted at DiaSorin, Saluggia Italy, to evaluate the performance of the ETI-MAK-2 PLUS assay on the Biochem Immunosystems Labotech/ETI-LAB, an automated microplate processing instrument, compared to the manual analysis. The Paul-Ehrlich-Institut (PEI) Standard, 12 serum samples near the ETI-MAK-2 PLUS cutoff and samples from the clinical trials (32 suspected hepatitis B patients and 8 apparently healthy adults) were tested in parallel manually and on the Labotech.

Serial dilutions of the PEI Standard were prepared in fetal calf serum to obtain a panel ranging from high concentration to below the analytical sensitivity of the assay. The diluted Standard samples were tested in duplicate, one run per day for three days both manually and on the Labotech. Due to the requirement that assay cutoff be established for each plate, reproducibility was evaluated based on specimen absorbance-to-cutoff ratios (S/CO) rather than absolute absorbance values. The $95\%$ confidence intervals were established for the S/CO values of each point of the Standard-referenced curve and therefore the analytical endpoint sensitivity was defined (first dilution with $\mathrm{S / CO} > 1.1$ ). A graph summarizing these results is presented below:


The 12 samples near the cutoff were tested in triplicate, one run per day for three days both manually and on the Labotech. The samples from the clinical trials were tested in singlet in one run on one day, both manually and on Labotech. The mean, the standard deviation and the coefficient of variation (CV%) of the S/CO values were computed by the different components of variability for each of the tested specimens. A summary of the data is presented in the following table.

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| | Manual | | | Labotech/ETI-LAB | | |
| --- | --- | --- | --- | --- | --- | --- |
| Analytical Endpoint Sensitivity (0.1 PEI U/mL) | Mean | W/R %CVa | D/D %CV | Mean | W/R %CV | D/D %CV |
| S/CO [95% CI]b | 1.56 [1.29 – 1.82] | 11.8 | 15.1 | 1.84 [1.56 – 2.12] | 4.1 | 15.7 |
| 12 Cutoff Samples S/CO Range of mean S/CO | 1.21 0.91 – 1.44 | 11.2 | 7.9 | 1.43 1.11 – 1.80 | 4.0 | 13.0 |
| Clinical Samples: | | | | | | |
| Suspected Hepatitis B Range of S/CO | Negative: N/A (0/32) Equivocal: N/A (0/32) Positive: 23.9 – >39 (32/32) | | | Negative: N/A (0/32) Equivocal: N/A (0/32) Positive: 35.5 – >62 (32/32) | | |
| Healthy Adults Range of S/CO | Negative: 0.24 – 0.64 (8/8) Equivocal: N/A (0/8) Positive: N/A (0/8) | | | Negative: 0.08 – 0.46 (8/8) Equivocal: N/A (0/8) Positive: N/A (0/8) | | |

$^{\mathrm{a}}$ %CVs were calculated using specimen absorbance-to-cutoff ratios (S/CO) which normalized the data plate-to-plate
$^{\mathrm{b}}$ 95% CI = 95% Confidence Interval; W/R = within-run; D/D = day-to-day

No reproducibility testing with the Labotech instrument was conducted. As part of the conditions of approval agreement, DiaSorin will provide FDA results from a reproducibility study using the Labotech instrument. Until that condition is met, the following statement will be placed in the labeling:

"Assay reproducibility using the Labotech has not been established. If the Labotech is used, the user should establish appropriate assay reproducibility in accordance with NCCLS EP5-A, Evaluation of Precision Performance of Clinical Chemistry Devices."

# 9.2. Analytical Sensitivity

The analytical sensitivity of the assay (the smallest quantity of analyte that can be distinguished from background) was evaluated using single point serial dilutions of a standard preparation from the Paul-Ehrlich-Institut (PEI). The analytical sensitivity of the assay (last positive dilution) was determined to be 0.1 PEI U/mL (Mean Signal-to-Cutoff Ratio = 1.56; 95% Confidence Interval = 1.29 to 1.82).

# 9.3. Potential Cross-Reacting Substances

Serum samples were obtained from patients belonging to a number of different disease categories listed below. Of the 525 potential interfering samples, 477 (91%) were negative and 48 (9%) were positive by ETI-MAK-2 PLUS. Among the 48 positive samples, 12 were negative for HBeAg and IgM anti-HBc and negative for HBsAg on repeat testing; 36 were positive by reference testing or review of hepatitis B marker patterns for those samples. As expected, individuals infected by HDV are also infected with HBV. Disease was determined by serological testing, there is no guarantee that the associated antigen was present in the tested material.

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# Cross-Reactivity Study Results

| GROUP | N | ETI-MAK-2 PLUS Negative or Equivocal Samples | ETI-MAK-2 PLUS Positive Samples | % Confirmed Positive By Additional Testing |
| --- | --- | --- | --- | --- |
| Acute EBV infection | 16 | 13 | 3a | - |
| Acute CMV infection | 20 | 17 | 3a | - |
| Acute HSV infection | 10 | 10 | 0 | - |
| Acute Toxoplasma infection | 18 | 18 | 0 | - |
| Acute parvovirus B19 infection | 5 | 5 | 0 | - |
| HTLV-I/II infection | 50 | 47 | 3b | 100% (1/1) |
| Syphilis | 26 | 25 | 1a | - |
| HCV infection | 50 | 48 | 2a | - |
| HDV infection | 20 | 1 | 19 | 100% (19/19) |
| HIV infection | 50 | 50 | 0 | - |
| Acute HAV infection | 50 | 47 | 3 | 100% (3/3) |
| Past HAV infection | 50 | 44 | 6c | 100% (5/5) |
| Rheumatoid factor (RF) + | 40 | 40 | 0 | - |
| Autoimmune disease, including SLE | 30 | 30 | 0 | - |
| Autoimmune hepatitis | 5 | 5 | 0 | - |
| Myeloma | 20 | 20d | 0 | - |
| Hypergammaglobulinemia | 20 | 20 | 0 | - |
| Influenza vaccine | 5 | 5 | 0 | - |
| Elevated liver enzymes | 10 | 9 | 1 | 100% (1/1) |
| Non-viral liver disease | 30 | 23 | 7 | 100% (7/7) |
| TOTAL | 525 | 477 (91%) | 48 (9%) | 100% (36/36) |

a These samples were negative for HBeAg and IgM anti-HBc, and negative on repeat testing on ETI-MAK-2 PLUS.
b 2 samples were negative for HBeAg and IgM anti-HBc, and negative on repeat testing on ETI-MAK-2 PLUS.
c 1 sample was negative for HBeAg and IgM anti-HBc, and negative on repeat testing on ETI-MAK-2 PLUS; 5 samples were positive by reference testing.
d 1 sample was repeatedly equivocal and all other markers were negative.

A BLAST analysis [Basic Local Alignment Search Tool, National Center for Biotechnology Information, National Institutes of Health, http://www.ncbi.nlm.nih.gov/BLAST] was performed to determine if untested viral or bacterial proteins could potentially cross-react with the anti-HBs monoclonal antibodies used in this assay. No notable similarities in protein sequences were identified from these viral or bacterial proteins, suggesting that they should not cross-react in this assay.

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# 9.4. Interfering Substances

The ETI-MAK-2 PLUS assay was evaluated for interference by testing the substances identified in the table below. Testing was performed using matched pairs of negative donor serum and negative donor serum spiked with high-titer HBsAg samples to obtain a result near the cutoff. None of the compounds at the levels indicated were found to interfere with the clinical interpretation of the assay in serum. No interference was found with bilirubin in plasma (EDTA, heparin or citrate), testing for interference with hemoglobin and triolein was not performed in plasma.

| Compound | Concentration | |
| --- | --- | --- |
| Bilirubin | 0.18 mmol/L | 10 mg/dL |
| Hemoglobin | 0.06 mmol/L | 100 mg/dL |
| Triolein | 33.9 mmol/L | 3000 mg/dL |

The ETI-MAK-2 PLUS assay was also evaluated for possible interference from heterophilic anti-mouse antibodies (HAMA). A dilutional panel was used, consisting of 21 samples prepared from a stock pool of high positive human serum. The HAMA concentrations in the samples ranged from 0 to 2975.5 ng/mL, as determined by a HAMA ELISA. In a direct non-competitive assay, such as ETI-MAK-2 PLUS, interference would manifest as false negative results. No interference was seen in that all 21 dilutions were negative by the ETI-MAK-2 PLUS assay, including the HAMA negative panel member.

# 9.5. Stability Studies

## 9.5.1. Kit Stability

Stability studies were performed on 3 different ETI-MAK-2 PLUS kit lots. At specified intervals from time of kit release, performance of the kits was evaluated by testing the Calibrator, Negative and Positive Controls, HBsAg calibration curve (subtype ad and ay; Paul Ehrlich Institut [PEI] referenced), and Q.C. sera panel according to the instructions for use. The kit must meet established acceptance criteria. The obtained stability data demonstrate that the kit performance is acceptable for at least 6 months. On the basis of the stability results, a shelf life of 6 months has been established for the kit.

## 9.5.2. Working Enzyme Tracer Stability

The Enzyme Tracer was diluted with the Tracer Diluent to obtain the working Enzyme Tracer according to the instructions for use. After 7 days from dilution, the performance of the kit was evaluated, according to the instructions for use, testing various specimens with freshly prepared working Enzyme Tracer and the 7-days old working Enzyme Tracer. The kit must meet established acceptance criteria. The tests on the working Enzyme Tracer demonstrate that the performance of the kit is acceptable when the 7 day-diluted Enzyme Tracer is used. The working Enzyme Tracer can be used for one week if stored at 2-8 °C.

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## 9.5.3. Working Wash Buffer Stability

The Wash Buffer concentrate was diluted with deionized water according to the instruction for use to obtain the working Wash buffer. After 7 days from dilution, the performance of the kit was evaluated by testing various specimens with a freshly prepared working Wash buffer and the 7-days-old working Wash buffer, according to the instructions for use. The kit must meet established acceptance criteria. The tests on the working Wash Buffer demonstrate that the performance of the kit is acceptable when the 7 day-old working Wash Buffer is used. The working Wash Buffer can be used for one week if stored at 2-8 °C.

## 9.6. Common Reagents interchangeability Study

Studies were performed to demonstrate that the lots of some components included with ETI-MAK-2 PLUS kit and common to all kits of ETI-PLUS line (Wash Buffer, Chromogen/Substrate, Stop Solution), can be exchanged with other lots of the same component produced for the ETI-PLUS line (interchangeability). Three lots of Wash Buffer, Chromogen/Substrate and Stop Solution were combined with one lot of ETI-MAK-2 PLUS; the three combinations were then tested with various samples. Regression analysis was applied to the Optical Density’s of the samples. The regression analyses for the three studies gave slopes close to 1.0, with low intercepts and excellent correlation values. These results indicate that the use of different lots of Wash Buffer, Chromogen/Substrate and Stop Solution with the same ETI-MAK-2 PLUS lot gave equivalent results with samples distributed over the range of reactivity, confirming their interchangeability.

## 9.7. Reproducibility

Manual Assay: Intra-assay, inter-assay, inter-lot, and inter-site variability studies were carried out on the ETI-MAK-2 PLUS kit to test the variability within runs, between runs, between days, between kit lots, and between test sites. Variability was measured on a panel of ten sera that included negative, borderline, and positive samples. Three ETI-MAK-2 PLUS kit lots were tested at three independent test sites. Due to the requirement that assay cutoff be established for each plate, reproducibility was evaluated based on specimen absorbance to cutoff ratios (S/CO) rather than absolute absorbance values. The results of that study are tabulated below showing the assay reproducibility to be satisfactory.

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Clinical Site Reproducibility Study

| ID# | | # of Tests per Sample | Mean S/CO's | Within-run %CV* | Between-runs %CV | Between-lots %CV | Between-days %CV | Between-sites %CV | Total |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| S01 | High | 108 | 13.09 | 6.08 | 12.11 | 10.63 | 9.70 | 9.03 | 16.51 |
| S02 | High | 108 | 5.59 | 4.44 | 10.89 | 7.82 | 11.60 | 8.98 | 17.50 |
| S03 | Low | 108 | 3.19 | 4.51 | 11.45 | 12.04 | 7.76 | 9.33 | 18.66 |
| S04 | Low | 108 | 2.70 | 7.47 | 8.14 | 12.65 | 8.61 | 6.72 | 20.18 |
| S05 | Low | 108 | 3.67 | 7.81 | 8.17 | 7.24 | 9.71 | 8.04 | 14.83 |
| S06 | Low | 108 | 2.57 | 4.40 | 17.13 | 12.58 | 16.81 | 7.31 | 25.88 |
| S07 | Equiv | 108 | 1.33 | 5.37 | 13.09 | 10.89 | 14.11 | 15.62 | 29.05 |
| S08 | Equiv | 108 | 0.96 | 9.19 | 10.09 | 13.48 | 8.82 | 5.00 | 24.04 |
| S09 | Equiv | 108 | 1.01 | 15.66 | 13.89 | 16.93 | 20.65 | 3.66 | 32.00 |
| S10 | Neg | 108 | 0.22 | 15.71 | 29.33 | 30.67 | 59.12 | 21.18 | 104.13 |

* %CVs were calculated using specimen absorbance-to-cutoff ratios (S/CO) which normalized the data plate-to-plate

# 9.8. Plasma Reproducibility

A plasma reproducibility study was conducted at DiaSorin, Saluggia Italy, to evaluate the performance of the manual ETI-MAK-2 PLUS assay on serum versus a variety of plasma types. The plasma types evaluated were citrate, heparin and EDTA. Sample sets of matched serum/multiple plasma were used in the study. A sample set was prepared by spiking the same high-positive sample into each of the matrices (serum and plasmas) resulting in a total of four specimens per set around the cutoff. Several high-positive samples were used in the preparation of the 12 different near-cutoff sample sets. Six matched serum/multiple plasma samples sets were tested in triplicate in each run; thus there were two runs per day for three days, all tested in a manual mode. Due to the requirement that assay cutoff be established for each plate, reproducibility was evaluated based on specimen absorbance to cutoff ratios (S/CO) rather than absolute absorbance values. The mean, the standard deviation and the coefficient of variation $(\mathrm{CV}\%)$ of the S/CO values were computed by the different components of variability for each of the tested specimens. The $95\%$ confidence intervals were established for the S/CO values of all serum samples and each plasma type. A summary of the data is presented in the following table. The study supports the use of plasma specimens in the ETI-MAK-2 PLUS assay.

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| | Serum | Citrate | Heparin | EDTA |
| --- | --- | --- | --- | --- |
| Mean S/CO | 0.91 | 0.92 | 0.87 | 0.91 |
| 95% CI* | [0.87-0.94] | [0.88-0.96] | [0.83-0.91] | [0.88-0.95] |
| W/R %CV** | 6.3% | 6.9% | 7.1% | 7.8% |
| D/D %CV | 8.1% | 9.6% | 6.7% | 8.0% |
| Total %CV | 9.9% | 11.6% | 9.6% | 10.0% |
| Between matrix %CV: 8.5% | | | | |
| Across matrix total %CV: 13.1% | | | | |

* 95% CI = 95% Confidence Interval; W/R = within-run; D/D = day-to-day
** %CVs were calculated using specimen absorbance-to-cutoff ratios (S/CO) which normalized the data plate-to-plate

# 9.9. Acute Serial Seroconversion Panels

One hundred twenty-four (124) archived serial samples from nine individuals were tested for the appearance of HBsAg. Most (8/9) of these individuals were defined as being acutely infected by the appearance of HBsAg and HBeAg with the subsequent appearance of IgM anti-HBc, total anti-HBc, anti-HBe, and anti-HBs. One individual had detectable HBsAg, but did not have detectable HBeAg in any specimen. However, this individual did seroconvert for anti-HBe.

The specimens were collected from individuals undergoing plasmapheresis for further manufacturing purposes. Three individuals were found to be infected with HBV during the first plasmapheresis and others became infected with HBV during subsequent plasmapheresis. It is unknown how long these three initially HBsAg reactives were infected prior to the first plasmapheresis. All nine individuals underwent sequential plasmapheresis after becoming HBV infected. However, the timing of subsequent plasmapheresis varied from individual to individual. The specimens draw times were normalized to represent the day that HBsAg was first detected by an FDA-licensed assay as day 0. Draw days ranged from day 0 (HBsAg first detected) through day 355 post-day 0. Since all panels did not contain the same draw day, sample results were grouped within day intervals (e.g., days 0, 1-10, 11-20, etc., representing days since first detection of HBsAg).

The results are summarized in the following table and graph. In the graph below the pattern for the reference HBsAg percent reactive has been overlaid for reference. The reference assay and the ETI-MAK-2 PLUS assay were very similar in performance when detecting HBsAg in seroconversion panels.

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| Day Range | Number Specimens | DiaSorin HBsAg Reactive | % Positive |
| --- | --- | --- | --- |
| 0 | 9 | 7 | 77.8% |
| 1-10 | 10 | 10 | 100.0% |
| 11-20 | 13 | 10 | 76.9% |
| 21-30 | 9 | 6 | 66.7% |
| 31-40 | 10 | 7 | 70.0% |
| 41-50 | 6 | 2 | 33.3% |
| 51-60 | 10 | 3 | 30.0% |
| 61-70 | 9 | 4 | 44.4% |
| 71-80 | 6 | 3 | 50.0% |
| 81-90 | 9 | 4 | 44.4% |
| 91-100 | 10 | 3 | 30.0% |
| 101-110 | 6 | 1 | 16.7% |
| 111-120 | 4 | 1 | 25.0% |
| 121-130 | 4 | 1 | 25.0% |
| 131-140 | 3 | 0 | 0.0% |
| 141-150 | 2 | 1 | 50.0% |
| 151-160 | 0 | 0 | NA |
| 161-170 | 1 | 0 | 0.0% |
| 171-180 | 0 | 0 | NA |
| 181-190 | 1 | 0 | 0.0% |
| 191-200 | 1 | 0 | 0.0% |
| 355 | 1 | 0 | 0.0% |


# 9.10. High Risk Population

Single repository samples belonging to high-risk populations (66 hemodialyzed patients, 148 hemophiliacs, 150 IV drug users) were tested with the DiaSorin ETI-MAK-2 PLUS assay to determine frequency of positive results in that population. The group was $12\%$ (42/364) female, $69\%$ (252/364) male, and $19\%$ (70/364) unspecified, with ages ranging from 19 to 87 years old. No geographical locations were specified. The table below summarizes the ETI-MAK-2 PLUS results. The data in the table represent the number of specimens and prevalence rate in each high risk population.

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High Risk Population

| Population | Frequency of Positive Results
(# Positive/Total # Samples) |
| --- | --- |
| IV drug users | 17/150 = 11.3% (2 equivocal) |
| Hemophiliacs | 12/148 = 8.1% |
| Hemodialysis patients | 0/66 = 0.0% |
| TOTAL | 29/364 = 8.0% (2 equivocal) |

9.11. Expected Values Study

The 236 prospective patient samples used in the expected values study for the ETI-MAK-2 PLUS assay to detect HbsAg were from patients who were sent to the laboratory for HBV testing. Of those, 100 (42%) were frozen and 136 (58%) were fresh. The patients represented Florida, Georgia, Pennsylvania, California, Utah, and the southeastern US. The group was 69% (162/236) female, 29% (68/236) male, and 2% (6/236) unspecified; the ethnicity of the patients was unspecified. The ages ranged from 5 to 88 years old, with 6 samples unspecified. The prevalence rate for HBsAg in patients who were sent to the laboratory for HBV testing was 10%.

The table below summarizes the percent ETI-MAK-2 PLUS positive and negative results by gender and age range. There were 6 samples for which gender and age were not reported; they were all positive. There were 6 other samples for which age was not reported, two were from females and four were from males; all were negative. These 12 results were not included in the table.

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Expected Values Summary

| | DiaSorin ETI-MAK-2 PLUS | | | | | | | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| | | + | | - | | E* | | TOTAL |
| Age Range | Gender | n | % | N | % | n | % | |
| 0-9 | F | 0 | 0 | 2 | 100 | 0 | 0 | 2 |
| | M | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
| 10-19 | F | 1 | 6 | 16 | 94 | 0 | 0 | 17 |
| | M | 1 | 50 | 1 | 50 | 0 | 0 | 2 |
| 20-29 | F | 3 | 6 | 48 | 94 | 0 | 0 | 51 |
| | M | 4 | 31 | 9 | 69 | 0 | 0 | 13 |
| 30-39 | F | 1 | 2 | 48 | 98 | 0 | 0 | 49 |
| | M | 3 | 18 | 14 | 82 | 0 | 0 | 17 |
| 40-49 | F | 3 | 15 | 16 | 80 | 1 | 5 | 20 |
| | M | 2 | 14 | 12 | 86 | 0 | 0 | 14 |
| 50-59 | F | 1 | 20 | 4 | 80 | 0 | 0 | 5 |
| | M | 1 | 13 | 7 | 88 | 0 | 0 | 8 |
| 60-69 | F | 1 | 33 | 2 | 67 | 0 | 0 | 3 |
| | M | 0 | 0 | 2 | 100 | 0 | 0 | 2 |
| 70-79 | F | 1 | 10 | 9 | 90 | 0 | 0 | 10 |
| | M | 0 | 0 | 5 | 100 | 0 | 0 | 5 |
| 80-89 | F | 1 | 33 | 2 | 67 | 0 | 0 | 3 |
| | M | 0 | 0 | 3 | 100 | 0 | 0 | 3 |
| TOTAL | | 23 | 10 | 200 | 89 | 1 | 1 | 224 |

*E = equivocal result

## 10. Summary of Clinical Studies

### 10.1. Clinical Sample Testing

#### 10.1.1. Prospective Samples

A study of 136 prospective specimens was conducted. These specimens represented individuals who were sent to the laboratory for hepatitis testing. Specimens were collected at a reference laboratory and assayed at the California clinical trial site. The patients were 86% (117/136) female and 14% (19/136) male. The ages ranged from 5 to 77 years old, with three specimens not specified.

The study (testing) sites were blinded to the previous specimen categorization. All testing was performed by the manual ETI-MAK-2 PLUS procedure. Specimens were characterized by testing with six HBV serological markers (HBsAg, HBeAg, IgM anti-HBc, total anti-HBc, anti-HBe, anti-HBs) using FDA-licensed or approved assays. Testing with these assays followed the FDA-licensed or approved labeling, including confirmation by neutralization of repeatably reactive HBsAg samples.

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# Results by Specimen Classification

After study completion all samples were assigned a specimen classification based on the patterns of the six HBV serological markers established by the reference assays. Based on these serological marker patterns, the samples were categorized into the HBV classifications described in the table below. There were five unique HBV marker patterns observed in the ETI-MAK-2 PLUS prospective clinical studies.

| Characterization Based On Single Point Specimen | HBsAg | HbeAg | IgM anti-HBc | Total anti-HBc | anti-HBe | anti-HBs | n |
| --- | --- | --- | --- | --- | --- | --- | --- |
| Chronic Infection | + | - | - | + | + | - | 1 |
| Recovery | - | - | - | + | + | + | 2 |
| Past Infection | - | - | - | + | - | + | 4 |
| | - | - | - | + | - | - | 4 |
| HBV Vaccine Response | - | - | - | - | - | + | 38 |
| Not Previously Infected with HBV | - | - | - | - | - | - | 87 |

Based on the above classifications the ETI-MAK-2 PLUS HBsAg results for the prospective samples were compared to a reference assay's HBsAg results. The following table shows this comparison and percent agreement with 95% confidence intervals with the reference HBs results.

Prospective Samples Comparison

| Serological Classification | Reference HBsAg^{a} | | | TOTAL |
| --- | --- | --- | --- | --- |
| | - | | + | |
| | ETI-MAK-2 PLUS | | ETI-MAK-2 PLUS | |
| | - | E^{b} | + | |
| Chronic infection | 0 | 0 | 1 | 1 |
| Recovery | 2 | 0 | 0 | 2 |
| Past infection | 8 | 0 | 0 | 8 |
| HBV vaccine response | 38 | 0 | 0 | 38 |
| Not previously infected | 86 | 1 | 0 | 87 |
| Grand Total | 134 | 1 | 1 | 136 |

a Result of initially repeatedly reactive and neutralization testing.
b Equivocal results

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# Prospective Samples Agreement Rates

| Chronic Infection | Positive agreement with reference assay results = 100% (1/1)
95% CI = 2.5 to 100%
Negative agreement with reference assay results = NA (0/0)
95% CI = NA |
| --- | --- |
| Recovery | Positive agreement with reference assay results = NA (0/0)
95% CI = NA
Negative agreement with reference assay results = 100% (2/2)
95% CI = 15.8 to 100% |
| HBV Vaccine Response | Positive agreement with reference assay results = NA (0/0)
95% CI = NA
Negative agreement with reference assay results = 100% (38/38)
95% CI = 90.8 to 100% |
| Past Infection | Positive agreement with reference assay results = NA (0/0)
95% CI = NA
Negative agreement with reference assay results = 100% (8/8)
95% CI = 63.1 to 100% |
| Not Previously Infected | Positive agreement with reference assay results = NA (0/0)
95% CI = NA
Negative agreement with reference assay results = 98.9% (86/87)
95% CI = 93.8 to 99.9% |

## 10.1.2. Retrospective Samples

Retrospective studies were carried out at three clinical laboratories in the United States (California, Missouri, and Minnesota) and at DiaSorin (Italy) to assess the performance of the ETI-MAK-2 PLUS assay in detecting HBsAg. The study set included 650 frozen repository samples (the majority of which were purchased from commercial vendors) from the following populations:

- patients with chronic hepatitis B infection (HBsAg positive for greater than six months) - 111 frozen repository samples;
- patients with serologically diagnosed hepatitis B infection (acute, chronic, asymptomatic, convalescent, etc.) - 82 frozen repository samples;
- patients sent to the laboratory for hepatitis B testing - 100 frozen repository samples;
- a general hospital patient population - 357 frozen repository samples.

The specimens represented Midwestern (2%), Southeastern (25%), Western (13%), and Northeastern US (2%), outside of the US (1%), and unspecified (57%). The group was 44% (287/650) female, 42% (270/650) male, and 14% (93/650) unspecified. Approximately 13% (84/650) were Caucasian, 4% (27/650) were African American, < 1% (5/650) were Hispanic, < 1% (3/650) were Asian, and 82% (531/650) were unspecified. The ages ranged from 5 to 98 years old, with 131 specimens not specified.

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The study (testing) sites were blinded to the previous specimen categorization. All testing was performed by the manual ETI-MAK-2 procedure. Specimens were characterized by testing with six HBV serological markers (HBsAg, HBeAg, IgM anti-HBc, total anti-HBc, anti-HBe, anti-HBs) using FDA-licensed or approved assays. Testing with these assays followed the FDA-licensed or approved procedure with the exception of the HBsAg assay at two of the three sites. At these sites, the majority of specimens that were initially HBsAg-positive were repeated in duplicate, however the repeatedly reactive specimens were not confirmed by the licensed HBsAg confirmation assay at the two sites. Therefore, true HBsAg result was determined in one of three ways: 1) confirmed by reference assay neutralization during clinical trials, 2) based on a statement by the attending physician that HBsAg was positive for greater than 6 months, or 3) information provided by the vendor regarding confirmatory testing performed at their location or by the material source facility.

## Results by Specimen Classification

After study completion all samples were assigned a specimen classification based on the patterns of the six HBV serological markers established by the reference assays. Based on these serological marker patterns, the samples were categorized into the HBV classifications described in the table below. There were 35 unique HBV marker patterns observed in the ETI-MAK-2 PLUS retrospective clinical studies.

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| Characterization Based On Single Point Specimen | HBsAg | HBeAg | IgM anti-HBc | Total anti-HBc | anti-HBe | anti-HBs | n |
| --- | --- | --- | --- | --- | --- | --- | --- |
| Acute Infection | + | + | + or I* | + | - | - | 52 |
| | + | - | + or I | + | + | - | 4 |
| | + | - | - | - | - | - | 2 |
| | + | + | - | - | - | - | 2 |
| Chronic Infection | + | - | - | + | + | - | 82 |
| | + | + | - | + | - | - | 21 |
| | + | - | - | + | - or I | - | 23 |
| | + | + | + | + | - | + | 4 |
| | + | + | - or I | + | - | + | 2 |
| | + | - | - | + | + | + | 2 |
| | + | + | - | + | + or I | + | 2 |
| | + | + | + | + | + | + | 1 |
| | + | + | - | + | + | - | 1 |
| | + | - | - | + | - | + | 1 |
| Recovery | - | - | - | + | + or I | + | 40 |
| | - | - | - | + | + | - | 6 |
| | - | - | + | + | + | - | 2 |
| | - | - | + or I | + | + | + | 2 |
| Past Infection | - | - or I | - | + | - | + | 12 |
| | - | - | - | + | - | - | 9 |
| HBV Vaccine Response | - | - | - | - | - | + | 20 |
| Not Previously Infected with HBV | - | - | - | - | - | - | 343 |
| Uninterpretable | - | + or I | - | - | - | - | 13 |
| | - | + | - | + | - | + | 2 |
| | - | + | - | + | + | + | 1 |
| | - | I | - | + | - | - | 1 |

*I = indeterminate result

Based on the above classifications the ETI-MAK-2 PLUS HBsAg results for the retrospective samples were compared to a reference assay's HBsAg results as determined by the methods described above. The following tables show this comparison and percent agreement with $95\%$ confidence intervals with the reference HBsAg results. The data are presented in three tables based on the means of determining true HBsAg results.

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# Retrospective Samples Comparison

True HBsAg Result Based on Statement from Physician

| Reference Serology Classification | Reference HBsAg | | TOTAL |
| --- | --- | --- | --- |
| | - | + | |
| | ETI-MAK-2 PLUS | ETI-MAK-2 PLUS | |
| | - | + | |
| Acute infection | 0 | 26 | 26 |
| Chronic infection | 0 | 61 | 61 |
| Recovery | 1 | 0 | 1 |
| Not previously infected | 1 | 0 | 1 |
| Grand Total | 2 | 87 | 89 |

# Retrospective Samples Agreement Rate

Acute Infection
Positive agreement with reference assay results = 100% (26/26)
95% CI = 86.8 to 100%
Negative agreement with reference assay results = NA (0/0)
95% CI = NA

Chronic Infection
Positive agreement with reference assay results = 100% (61/61)
95% CI = 94.1 to 100%
Negative agreement with reference assay results = NA (0/0)
95% CI = NA

Recovery
Positive agreement with reference assay results = NA (0/0)
95% CI = NA
Negative agreement with reference assay results = 100% (1/1)
95% CI = 2.5 to 100%

Not Previously Infected
Positive agreement with reference assay results = NA (0/0)
95% CI = NA
Negative agreement with reference assay results = 100% (1/1)
95% CI = 2.5 to 100.0%

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Retrospective Samples Comparison

True HBsAg Result Based on Confirmation by Neutralization during Clinical Trials

| Reference Serology Classification | Reference HbsAg | | | | TOTAL |
| --- | --- | --- | --- | --- | --- |
| | - | | + | | |
| | ETI-MAK-2 PLUS | | ETI-MAK-2 PLUS | | |
| | - | + | - | + | |
| Acute infection | 0 | 0 | 0 | 25 | 25 |
| Chronic infection | 0 | 0 | 1 | 47 | 48 |
| Recovery | 16 | 0 | 0 | 0 | 16 |
| Past infection | 6 | 0 | 0 | 0 | 6 |
| HBV vaccine response | 6 | 0 | 0 | 0 | 6 |
| Not previously infected | 110 | 3 | 0 | 0 | 113 |
| Uninterpretable | 5 | 2 | 0 | 0 | 7 |
| Grand Total | 143 | 5 | 1 | 72 | 221 |

Retrospective Samples Agreement Rate

| Acute Infection | Positive agreement with reference assay results = 100% (25/25)95% CI = 86.3 to 100%Negative agreement with reference assay results = NA (0/0)95% CI = NA |
| --- | --- |
| Chronic Infection | Positive agreement with reference assay results = 97.9% (47/48)95% CI = 88.9 to 99.9%Negative agreement with reference assay results = NA (0/0)95% CI = NA |
| Recovery | Positive agreement with reference assay results = NA (0/0)95% CI = NANegative agreement with reference assay results = 100% (16/16)95% CI = 79.4 to 100% |
| HBV Vaccine Response | Positive agreement with reference assay results = NA (0/0)95% CI = NANegative agreement with reference assay results = 100% (6/6)95% CI = 54.1 to 100.0% |
| Past Infection | Positive agreement with reference assay results = NA (0/0)95% CI = NANegative agreement with reference assay results = 100% (6/6)95% CI = 54.1 to 100% |
| Not Previously Infected | Positive agreement with reference assay results = NA (0/0)95% CI = NANegative agreement with reference assay results = 97.3% (110/113)95% CI = 92.4 to 99.4% |
| Uninterpretable | Positive agreement with reference assay results = NA (0/0)95% CI = NANegative agreement with reference assay results = 71.4% (5/7)95% CI = 29.0 to 96.3% |

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Retrospective Samples Comparison
True HBsAg Result Based on Vendor Information

| Reference Serology Classification | Reference HbsAg | | | | TOTAL |
| --- | --- | --- | --- | --- | --- |
| | - | | + | | |
| | ETI-MAK-2 PLUS | | ETI-MAK-2 PLUS | | |
| | - | + | + | E* | |
| Acute infection | 0 | 0 | 6 | 0 | 6 |
| Chronic infection | 0 | 0 | 22 | 1 | 23 |
| Recovery | 33 | 0 | 0 | 0 | 33 |
| Past infection | 15 | 0 | 0 | 0 | 15 |
| HBV vaccine response | 13 | 1 | 0 | 0 | 14 |
| Not previously infected | 222 | 7 | 0 | 0 | 229 |
| Uninterpretable | 9 | 1 | 0 | 0 | 10 |
| Grand Total | 292 | 9 | 28 | 1 | 330 |

* Equivocal results

Retrospective Samples Agreement Rate

| Acute Infection | Positive agreement with reference assay results = 100% (6/6) |
| --- | --- |
| | 95% CI = 54.1 to 100% |
| | Negative agreement with reference assay results = NA (0/0) |
| | 95% CI = NA |
| Chronic Infection | Positive agreement with reference assay results = 100% (22/22) |
| | 95% CI = 84.6 to 100% |
| | Negative agreement with reference assay results = NA (0/0) |
| | 95% CI = NA |
| Recovery | Positive agreement with reference assay results = NA (0/0) |
| | 95% CI = NA |
| | Negative agreement with reference assay results = 100% (33/33) |
| | 95% CI = 89.4 to 100% |
| HBV Vaccine Response | Positive agreement with reference assay results = NA (0/0) |
| | 95% CI = NA |
| | Negative agreement with reference assay results = 92.9% (13/14) |
| | 95% CI = 66.1 to 99.8% |
| Past Infection | Positive agreement with reference assay results = NA (0/0) |
| | 95% CI = NA |
| | Negative agreement with reference assay results = 100% (15/15) |
| | 95% CI = 78.2 to 100% |
| Not Previously Infected | Positive agreement with reference assay results = NA (0/0) |
| | 95% CI = NA |
| | Negative agreement with reference assay results = 96.9% (222/229) |
| | 95% CI = 93.8 to 98.8% |
| Uninterpretable | Positive agreement with reference assay results = NA (0/0) |
| | 95% CI = NA |
| | Negative agreement with reference assay results = 90.0% (9/10) |
| | 95% CI = 55.5 to 99.7% |

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# 10.1.3. Samples from Pregnant Women:

Single samples collected from pregnant women (154 prospective samples and 410 retrospective samples) were tested with both DiaSorin and reference HBsAg assays. Positive results from the retrospective samples were confirmed by reference method neutralization at DiaSorin Italy after completion of the trials. Nonreactive results from the prospective samples were verified by testing for HBeAg, anti-HBe, total anti-HBc and IgM anti-HBc at the trial site. All nonreactive specimens were HBeAg and IgM anti-HBc nonreactive confirming the nonreactive HBsAg. The one positive sample from the prospective population was repeat tested for HBsAg and confirmed by reference method neutralization at the trial site. The table below compares the ETI-MAK-2 PLUS results with the HBsAg reference assay. The data in the table represent the number of specimens in each group. Equivocal results by the ETI-MAK-2 PLUS were repeated per the insert instructions, if sample volumes permitted and the repeat results used in the calculation.

Pregnant Women – Samples Comparison

| Group | Reference HBsAg | | | | | Total |
| --- | --- | --- | --- | --- | --- | --- |
| | - | | + | | | |
| | ETI-MAK-2 PLUS | | ETI-MAK-2 PLUS | | | |
| | - | + | - | + | E* | |
| Prospective Samples | 154 | 0 | 0 | 0 | 0 | 154 |
| Retrospective Samples | 378 | 12 | 1 | 17 | 2 | 410 |
| Total | 532 | 12 | 1 | 17 | 2 | 564 |

* Equivocal results

Pregnant Women Prospective Samples Agreement Rate

Percent Positive Agreement = 0.% (0/0) 95% CI = NA

Percent Negative Agreement = 100.% (154/154) 95% CI = 97.6 to 100.

Pregnant Women Retrospective Samples Agreement Rate

Percent Positive Agreement = 85.0 (17/20) 95% CI = 62.1 to 96.8%

Percent Negative Agreement = 96.9% (378/390) 95% CI = 94.7 to 98.4

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# 11. Conclusions Drawn from Studies

The study data demonstrates that acceptable performance is obtained with the DiaSorin ETI-MAK-2 PLUS assay when testing specimens collected in serum and plasma. There appeared to be no gender bias in the selection ratio or any difference in the safety and effectiveness based on gender. The DiaSorin assay shows acceptable within-run, between-run, between-day, site-to-site, and lot-to-lot reproducibility. The quality control procedures described in the package insert are appropriate to assure accurate assay performance. The data from this study provide reasonable assurance that the DiaSorin ETI-MAK-2 PLUS assay is safe and effective for its stated purpose when used as instructed in the package insert. The DiaSorin ETI-MAK-2 PLUS HBsAg assay can be stored up to 6 months weeks at 2-8°C.

---

## Benefit/Risk

The submitted studies have shown that the DiaSorin ETI-MAK-2 PLUS assay, when compared to reference laboratory procedures, has a similar ability to detect the presence of HBsAg in specimens from individuals acutely and chronically infected with HBV. The rate of false positivity and false negativity are within acceptable limits compared to the reference assay. It has been shown that the device has no demonstrable cross-reactivity with viruses or organisms that may cause clinical hepatitis. Therefore, the devices should benefit the physician in the diagnosis of HBV associated and non-associated hepatitis. The devices will also aid in the identification of pregnant women infected with HBV. Therefore it is reasonable to conclude that the benefits of use of the device for the target population outweigh the risk of illness or injury when used as indicated when used accordance with the directions for use.

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# 12. Panel Recommendations

The Microbiology Advisory Panel met on January 20, 2000, to consider the safety and effectiveness of the ETI-MAK-2 PLUS assay. The Panel recommended approval subject to the following conditions.

- Conduct additional studies for the immunity claim by testing individuals immediately after receiving the complete series of three vaccinations with the hepatitis B virus vaccines and three to nine months later.
- Provide more data on acute/chronic infections in high-risk populations such as those individuals that are infected with HIV, sexually transmitted diseases, and those patients that are immunosuppressed.
- Collect more data on patients meeting the standard definition for chronicity, i.e., > 6 months of infection.

---

# 13. CDRH Decision

CDRH concurred with the Panel’s recommendation. DiaSorin Inc. has provided some additional data to address some of the Panel’s issues and those issues not fully resolved were addressed with labeling restrictions and the requirement of postapproval studies. The two postapproval studies were:

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- Within 6 months of this approval, DiaSorin should submit a reproducibility study for the Biochem Immunosystems Labotech/ETI-Lab automated instrument.

- To address the concerns made by the Panel regarding the retrospective nature of the clinical studies, within 2 years of this approval, DiaSorin should submit the results of an additional prospective clinical study. We suggest that this study involve individuals that may be considered representative of an U.S. population, i.e., similar prevalence of HBV disease and serotypes.

The applicant’s manufacturing facility was found to be in compliance with the Quality Systems Regulation (21 CFR 820).

CDRH issued an approval order on March 30, 2001.

## 14. Approval Specifications

Directions for use: See Labeling

Conditions of Approval: CDRH Approval of this PMA is subject to full compliance with the conditions described in the approval order.

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