**Summary of Safety and Effectiveness**

1.  **General Information**

    1.  Device Generic Name: Immunoassay for hepatitis Be antigen

    2.  Device Trade Name: ETI-EBK PLUS

    3.  Applicant's Name and Address:

######## DiaSorin

> Via Crescentino

Saluggia (VC) 13040, Italy

4.  U.S. Representative:

> Sienna Partners, LLC
>
> P.O. Box 103

Baldwin, MD 21013

5.  PMA Number: P990043

6.  Date of Panel Recommendation: January 20, 2000

7.  Date of Notice of Approval to the Applicant: February 5, 2001

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2.  **Indications For Use**

> ETI-EBK PLUS is an *in vitro* enzyme immunoassay (EIA) intended for
> use in the qualitative detection of hepatitis Be antigen (HBeAg) in
> human serum or plasma (EDTA, citrate or heparin). The ETI-EBK PLUS is
> intended for manual use and with the Biochem Immunosystems
> Labotech/ETI-Lab automated instrument.
>
> The detection of HBeAg is indicative of a laboratory diagnosis for
> active HBV replication, usually correlating with viremia. A reactive
> test is presumptive laboratory evidence for infection with hepatitis B
> virus (HBV). Further HBV serological marker testing is required to
> define the specific disease-state. The ETI-EBK PLUS assay's
> performance has not been established for the monitoring of HBV disease
> or therapy.

3.  **Device Description**

    1.  Principle of The Assay

> ETI-EBK PLUS uses monoclonal antibodies to hepatitis Be antigen 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 HBeAg and an enzyme tracer
> containing horseradish-peroxidase-labeled mouse monoclonal antibodies
> to HBeAg. The monoclonal antibodies are of the IgG1-k class and are
> directed to p16, the secreted form of HBeAg.
>
> In the assay procedure, patient specimens and controls are incubated
> with an incubation buffer in antibody-coated microwells. If HBeAg is
> present in a specimen or control, it binds to the antibody. 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 HBeAg, the bound enzyme (horseradish peroxidase)
> chemically reduces the substrate peroxide, which concurrently oxidizes
> the chromogen tetramethylbenzidine (TMB) to a blue color. The blue
> color turns to yellow after addition of the stop solution. If a sample
> does not contain HBeAg, 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 directly related to the presence of HBeAg.
> Absorbance value readings for patient specimens are compared to a
> cutoff value determined from the mean absorbance value of the
> calibrator.

3.2. Kit Configuration and Components

For detection of HBeAg, the ETI-EBK PLUS system is comprised of the
following:

> Coated Strips
>
> Microwells coated with mouse monoclonal antibodies to HBeAg.
>
> Enzyme Tracer
>
> Horseradish peroxidase-labeled mouse monoclonal antibody to HBeAg,
> buffer, protein stabilizers.
>
> Preservative: 0.2% ProClin 300.
>
> Tracer Diluent
>
> Buffer, protein stabilizers.
>
> Preservative: 0.2% ProClin 300.
>
> Calibrator (Human)
>
> Human serum/plasma non-reactive for all known HBV markers.
>
> Preservative: 0.2% ProClin 300.
>
> Negative Control (Human)
>
> Human serum/plasma non-reactive for all known HBV markers.
>
> Preservative: 0.2% ProClin 300.
>
> Positive Control
>
> HBeAg (recombinant DNA), 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
>
> 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-EBK PLUS assay are
> stated in the product labeling.

6.  **Alternative Practices and Procedures**

> Several methods are available for detecting HBeAg in human serum or
> plasma. Methods commonly used include immunodiffusion,
> electrophoresis, counter immunoelectrophresis, reverse passive
> hemagglutination, radioimmunoassay, and enzyme immunoassay.

7.  **Marketing History**

> The ETI-EBK-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.

9.  **Summary of Preclinical Studies**

    1.  Comparison of Labotech Instrumentation with the Manual Assay

> ![](media/image1.wmf){width="4.625in" height="3.1875in"}An instrument
> application study was conducted at DiaSorin, Saluggia Italy, to
> evaluate the performance of the ETI-EBK 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-EBK PLUS cutoff and
> samples from the clinical trials (29 suspected hepatitis B patients
> and 11 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 S/CO \> 1.1). A graph summarising 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.

+----------------------+----------+-----+-----+----------+----+-----+
|                      | Manual   |     |     | Labotech |    |     |
|                      |          |     |     | /ETI-LAB |    |     |
+======================+==========+=====+=====+==========+====+=====+
| Analytical Endpoint  | Mean     | W/R | D/D | Mean     | W  | D/D |
| Sensitivity\         |          | %CV | %CV |          | /R | %CV |
| (0.18 PEI U/mL)      |          | ^a^ |     |          | %  |     |
|                      |          |     |     |          | CV |     |
| > S/CO               |          |     |     |          |    |     |
| >                    |          |     |     |          |    |     |
| > \[95% CI\]^b^      |          |     |     |          |    |     |
+----------------------+----------+-----+-----+----------+----+-----+
|                      | 1.87     | 4.2 | 5.8 | 1.97     | 4  | 7.2 |
|                      |          |     |     |          | .0 |     |
|                      | \[1.75   |     |     | \[1.82   |    |     |
|                      | --       |     |     | --2.13\] |    |     |
|                      | 1.99\]   |     |     |          |    |     |
+----------------------+----------+-----+-----+----------+----+-----+
| 12 Cutoff Samples    | 0.96     | 7.1 | 5.8 | 1.09     | 5  | 3.7 |
| S/CO                 |          |     |     |          | .0 |     |
|                      | 0.74 --  |     |     | 0.84 --  |    |     |
| Range of S/CO        | 1.11     |     |     | 1.27     |    |     |
+----------------------+----------+-----+-----+----------+----+-----+
| Clinical Samples:    |          |     |     |          |    |     |
+----------------------+----------+-----+-----+----------+----+-----+
| Suspected Hepatitis  | N        |     |     | N        |    |     |
| B                    | egative: |     |     | egative: |    |     |
|                      | 0.00 --  |     |     | 0.06 --  |    |     |
| Range of S/CO        | 0.50     |     |     | 0.71     |    |     |
|                      | (8/29)\  |     |     | (8/29)\  |    |     |
|                      | Eq       |     |     | Eq       |    |     |
|                      | uivocal: |     |     | uivocal: |    |     |
|                      | 094      |     |     | N/A      |    |     |
|                      | (1/29)\  |     |     | (0/29)\  |    |     |
|                      | P        |     |     | P        |    |     |
|                      | ositive: |     |     | ositive: |    |     |
|                      | 6.46 --  |     |     | 1.15 --  |    |     |
|                      | \>51     |     |     | \>45     |    |     |
|                      | (20/29)  |     |     | (21/29)  |    |     |
+----------------------+----------+-----+-----+----------+----+-----+
| Healthy Adults       | N        |     |     | N        |    |     |
|                      | egative: |     |     | egative: |    |     |
| Range of S/CO        | -0.05 -- |     |     | 0.09 --  |    |     |
|                      | 0.43     |     |     | 0.44     |    |     |
|                      | (11/11)\ |     |     | (11/11)\ |    |     |
|                      | Eq       |     |     | Eq       |    |     |
|                      | uivocal: |     |     | uivocal: |    |     |
|                      | N/A      |     |     | N/A      |    |     |
|                      | (0/11)\  |     |     | (0/11)\  |    |     |
|                      | P        |     |     | P        |    |     |
|                      | ositive: |     |     | ositive: |    |     |
|                      | N/A      |     |     | N/A      |    |     |
|                      | (0/11)   |     |     | (0/11)   |    |     |
+----------------------+----------+-----+-----+----------+----+-----+

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

^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 was evaluated using single
> point serial dilutions of a standard preparation from the
> Paul-Ehrlich-Institut(PEI), Germany. The analytical sensitivity of the
> assay was determined to be 0.2 PEI U/mL.

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, 513 (98%) were negative and 12 (2%) were positive
by ETI-EBK PLUS. Among the 12 positive samples, 4 were negative for
HBsAg and IgM anti-HBc and negative for HBeAg on repeat testing; 7 were
positive by reference testing or review of hepatitis B marker patterns
for those samples. Disease was determined by serological testing, there
is no guarantee that the associated antigen was present in the tested
material. Interference testing with antigen in the described specimens
was not performed.

> The following table shows the summary of cross-reactivity tests:

  ------------------------------------------------------------------------
  DISEASE CATERGORY        N        ETI-EBK      ETI-EBK PLUS \% Confirmed
                                    PLUS\        Positive     Positive By
                                    Negative     Samples      Additional
                                    Samples                   Testing
  ------------------------ -------- ------------ ------------ ------------
  Acute EBV infection      16       16           0            \-

  Acute CMV infection      20       20           0            \-

  Acute HSV infection      10       10           0            \-

  Acute Toxoplasma         18       18           0            \-
  infection                                                   

  Acute Parvovirus B19     5        5            0            \-
  infection                                                   

  HTLV-I/II infection      50       48           2\*          \-

  Syphilis                 26       26           0            \-

  HCV infection            50       50           0            \-

  HDV infection            20       17           3            100% (3/3)

  HIV infection            50       49           1\*          \-

  Acute HAV infection      50       49           1            0% (0/1)

  Past HAV infection       50       48           2            100% (2/2)

  Rheumatoid factor (RF) + 40       40           0            \-

  Autoimmune disease       19       19           0            \-

  Systemic lupus           11       11           0            \-
  erythrematosus (SLE)                                        

  Autoimmune hepatitis     5        5            0            \-

  Myeloma                  20       19           1\*          \-

  Hypergammaglobulinemia   20       20           0            \-

  Influenza vaccine        5        5            0            \-

  Elevated liver enzymes   10       10           0            \-

  Non-viral liver disease  30       28           2            100%(2/2)

  **TOTAL**                525      513 (98%)    12 (2%)      88% (7/8)
  ------------------------------------------------------------------------

> \*These samples were negative for HBsAg and IgM anti-HBc, and negative
> on repeat testing on ETI-EBK PLUS.

## A BLAST (Basic Local Alignment Search Tool, National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health) analysis was performed to determine if untested viral or bacterial proteins could potentially cross-react with the anti-HBe monoclonal antibodies used in this assay. The only protein that showed some homology was the HBV core antigen. Additional cross-reactivity studies showed that the HBc antigen cross-reacted with the assay only when the monoclonal anti-HBe was present at approximately 83 times the concentration used in the assay. No notable similarities in protein sequences were identified from the other viral or bacterial proteins, suggesting that they should not cross-react in this assay.

######  9.4. Interfering Substances

DiaSorin ETI-EBK PLUS assay was evaluated for interference by testing
the following substances listed below. Testing was performed using
matched pairs of negative donor serum and negative donor serum spiked
with a high titer HBeAg 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              | 0.35 mmol/L 20 mg/dL                        |
|                        |                                             |
| Hemoglobin             | 0.06 mmol/L 100 mg/dL                       |
|                        |                                             |
| Triolein               | 33.9 mmol/L 3000 mg/dL                      |
+------------------------+---------------------------------------------+

> The ETI-EBK 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-EBK PLUS, interference would
> manifest as false positive results. No interference was seen in that
> all 21 dilutions were negative by the ETI-EBK PLUS assay, and
> supported by positive anti-HBe results.
>
> 9.5. Stability Studies

9.5.1. Kit Stability

> Stability studies were performed on 3 different ETI-EBK PLUS kit lots.
> At specified intervals from time of kit release, performance of the
> kits was evaluated testing the Calibrator, Negative and Positive
> Controls, HBeAg calibration curve (Paul Ehrlich Institut \[PEI\]
> referenced), and Q.C. sera panel according to the instructions for
> use. The kit must meet established acceptance criteria. 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.

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-EBK 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-EBK PLUS; the
> three combinations were then tested with various samples. Regression
> analysis was applied to the Optical Densities 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-EBK 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-EBK 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-EBK 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 reproducibility
> of the assay to be satisfactory.

####### Clinical Site Reproducibility Study

<table>
<colgroup>
<col style="width: 8%" />
<col style="width: 8%" />
<col style="width: 12%" />
<col style="width: 9%" />
<col style="width: 9%" />
<col style="width: 9%" />
<col style="width: 11%" />
<col style="width: 10%" />
<col style="width: 11%" />
<col style="width: 8%" />
</colgroup>
<tbody>
<tr class="odd">
<td>ID#</td>
<td></td>
<td><strong>Number of tests per sample</strong></td>
<td><p><strong>Mean</strong></p>
<p>S/CO</p></td>
<td>Within-run<br />
%CV*</td>
<td><p>Between-runs</p>
<p>%CV</p></td>
<td><p>Between-lots</p>
<p>%CV</p></td>
<td><p>Between-days</p>
<p>%CV</p></td>
<td><p>Between-sites</p>
<p>%CV</p></td>
<td>Total</td>
</tr>
<tr class="even">
<td>S01</td>
<td>High</td>
<td>108</td>
<td>20.03</td>
<td>6.37</td>
<td>6.29</td>
<td>10.07</td>
<td>7.30</td>
<td>5.14</td>
<td>14.47</td>
</tr>
<tr class="odd">
<td>S02</td>
<td>High</td>
<td>108</td>
<td>10.83</td>
<td>4.16</td>
<td>5.07</td>
<td>11.56</td>
<td>7.34</td>
<td>9.44</td>
<td>15.69</td>
</tr>
<tr class="even">
<td>S03</td>
<td>High</td>
<td>108</td>
<td>6.83</td>
<td>4.47</td>
<td>7.16</td>
<td>11.78</td>
<td>7.09</td>
<td>6.73</td>
<td>17.18</td>
</tr>
<tr class="odd">
<td>S04</td>
<td>Low</td>
<td>108</td>
<td>3.84</td>
<td>3.80</td>
<td>8.25</td>
<td>14.42</td>
<td>6.97</td>
<td>5.79</td>
<td>17.12</td>
</tr>
<tr class="even">
<td>S05</td>
<td>Equiv</td>
<td>108</td>
<td>0.80</td>
<td>6.08</td>
<td>6.79</td>
<td>8.51</td>
<td>6.38</td>
<td>3.79</td>
<td>13.03</td>
</tr>
<tr class="odd">
<td>S06</td>
<td>Low</td>
<td>108</td>
<td>2.61</td>
<td>5.35</td>
<td>5.99</td>
<td>9.72</td>
<td>7.92</td>
<td>9.80</td>
<td>14.35</td>
</tr>
<tr class="even">
<td>S07</td>
<td>Low</td>
<td>108</td>
<td>2.11</td>
<td>3.55</td>
<td>10.49</td>
<td>10.44</td>
<td>7.47</td>
<td>10.18</td>
<td>17.97</td>
</tr>
<tr class="odd">
<td>S08</td>
<td>Low</td>
<td>108</td>
<td>1.96</td>
<td>5.08</td>
<td>11.86</td>
<td>10.47</td>
<td>6.86</td>
<td>7.30</td>
<td>16.18</td>
</tr>
<tr class="even">
<td>S09</td>
<td>Equiv</td>
<td>108</td>
<td>1.67</td>
<td>6.27</td>
<td>11.85</td>
<td>10.47</td>
<td>4.04</td>
<td>4.04</td>
<td>19.19</td>
</tr>
<tr class="odd">
<td>S10</td>
<td>Neg</td>
<td>108</td>
<td>0.06</td>
<td>20.20</td>
<td>36.73</td>
<td>36.18</td>
<td>15.26</td>
<td>29.61</td>
<td>89.32</td>
</tr>
</tbody>
</table>

\* %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-EBK 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 (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-EBK PLUS assay.

  --------------- ---------- ------- ------------- -------------- -------------
                  SERUM              CITRATE       HEPARIN        EDTA

  Mean (S/CO)     0.99               0.99          1.02           1.04

  95% CI\*        \[0.95 -           \[0.95 --     \[0.98 --      \[1.00 --
                  1.03\]             1.03\]        1.06\]         1.08\]

  W/R %CV\*\*     5.9%               4.8%          5.4%           4.5%

  D/D %CV         9.3%               7.7%          7.2%           8.5%

  Total %CV       10.4%              8.6%          8.6%           8.9%

  Between matrix             7.9%                                 
  %CV                                                             

  Across matrix              11.7%                                
  total %CV                                                       
  --------------- ---------- ------- ------------- -------------- -------------

\* 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 {#acute-serial-seroconversion-panels .unnumbered}

One hundred twenty-four (124) archived serial samples from nine
individuals were tested for the appearance of HBeAg. Most (8/9) of these
individuals were defined as being acutely infected by the appearance of
HBsAg and HBeAg with the subsequence appearance of IgM anti-HBc, total
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 a FDA licensed assay as Day 0. For the remaining
specimens, 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. No specimen was
reactive for HBeAg by the DiaSorin assay after day 90. In the graph
below a dashed line for the reference HBsAg percent reactive has been
overlaid for reference.

Acute Serial Panel Results

> ![](media/image2.wmf)

+--------------+--------------+---------------------+------------------+
| > Day Range  | > Number     | > DiaSorin HBeAg    | > \% Positive    |
|              | > Specimens  | > Reactive          |                  |
+--------------+--------------+---------------------+------------------+
| 0            | 9            | 1                   | 11.1             |
+--------------+--------------+---------------------+------------------+
| 1-10         | 10           | 5                   | 50.0             |
+--------------+--------------+---------------------+------------------+
| 11-20        | 13           | 10                  | 76.9             |
+--------------+--------------+---------------------+------------------+
| 21-30        | 9            | 6                   | 66.7             |
+--------------+--------------+---------------------+------------------+
| 31-40        | 10           | 7                   | 70.0             |
+--------------+--------------+---------------------+------------------+
| 41-50        | 6            | 1                   | 16.7             |
+--------------+--------------+---------------------+------------------+
| 51-60        | 10           | 2                   | 20.0             |
+--------------+--------------+---------------------+------------------+
| 61-70        | 9            | 3                   | 33.3             |
+--------------+--------------+---------------------+------------------+
| 71-80        | 6            | 2                   | 33.3             |
+--------------+--------------+---------------------+------------------+
| 81-90        | 9            | 1                   | 11.1             |
+--------------+--------------+---------------------+------------------+
| 91-100       | 10           | 0                   | NA               |
+--------------+--------------+---------------------+------------------+
| 101-110      | 6            | 0                   | NA               |
+--------------+--------------+---------------------+------------------+
| 111-120      | 4            | 0                   | NA               |
+--------------+--------------+---------------------+------------------+
| 121-130      | 4            | 0                   | NA               |
+--------------+--------------+---------------------+------------------+
| 131-140      | 3            | 0                   | NA               |
+--------------+--------------+---------------------+------------------+
| 141-150      | 2            | 0                   | NA               |
+--------------+--------------+---------------------+------------------+
| 151-160      | 0            | 0                   | NA               |
+--------------+--------------+---------------------+------------------+
| 161-170      | 1            | 0                   | NA               |
+--------------+--------------+---------------------+------------------+
| 171-180      | 0            | 0                   | NA               |
+--------------+--------------+---------------------+------------------+
| 181-190      | 1            | 0                   | NA               |
+--------------+--------------+---------------------+------------------+
| 191-200      | 1            | 0                   | NA               |
+--------------+--------------+---------------------+------------------+
| 355          | 1            | 0                   | NA               |
+--------------+--------------+---------------------+------------------+

9.10. Expected Values Study

> The 236 prospective samples used in the expected values study for the
> DiaSorin ETI-EBK PLUS assay 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 and 31% (74/236) male; the ethnicity of the
> patients was unspecified. The ages ranged from 5 to 88 years old. The
> prevalence rate for HBeAg in patients who were sent to the laboratory
> for HBV testing was 7%.
>
> The Table below summarizes the percent DiaSorin ETI-EBK 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 samples for which age was not reported, 2 were from females and
> 4 were from males; all were negative. These 12 results were not
> included in the table.
>
> Expected Values Summary

  ------------------------------------------------------------------------------
                        DiaSorin                                  
                        ETI-EBK                                   
                        PLUS                                      
  ----------- --------- ---------- --------- -------- ----------- --------------
                        \+                   \-                   

  Age Range   Gender    n          \%        n        \%          TOTAL

  0-9         F         0          0%        2        100%        2

              M         0          NA        0        NA          0

  10-19       F         1          6%        16       94%         17

              M         1          50%       1        50%         2

  20-29       F         1          2%        50       98%         51

              M         3          23%       10       77%         13

  30-39       F         1          2%        48       98%         49

              M         1          6%        16       94%         17

  40-49       F         1          5%        19       95%         20

              M         1          7%        13       93%         14

  50-59       F         4          80%       1        20%         5

              M         1          13%       7        88%         8

  60-69       F         0          0%        3        100%        3

              M         0          0%        2        100%        2

  70-79       F         0          0%        10       100%        10

              M         0          0%        5        100%        5

  80-89       F         0          0%        3        100%        3

              M         0          0%        3        100%        3

  TOTAL                 15         7%        209      93%         224
  ------------------------------------------------------------------------------

10. **Summary of Clinical Studies**

    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 3 specimens not specified. The study (testing) sites
> were blinded to the previous specimen categorization. All testing was
> performed by the manual ETI-EBK 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.

# 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 5 unique HBV marker patterns
observed in the DiaSorin ETI-EBK PLUS prospective clinical studies.

  -------------------------------------------------------------------------------------------
  Characterization Based   HBsAg   HBeAg   IgM        Total      anti-HBe   anti-HBs   n
  On Single Point Specimen                 anti-HBc   anti-HBc                         
  ------------------------ ------- ------- ---------- ---------- ---------- ---------- ------
  Chronic Infection        \+      \-      \-         \+         \+         \-         1

  Recovery                 \-      \-      \-         \+         \+         \+         2

  Past Infection           \-      \-      \-         \+         \-         \+         4

                           \-      \-      \-         \+         \-         \-         4

  HBV Vaccine Response     \-      \-      \-         \-         \-         \+         38

  Not Previously Infected  \-      \-      \-         \-         \-         \-         87
  with HBV                                                                             
  -------------------------------------------------------------------------------------------

> Based on the above classifications the DiaSorin HBeAg results for the
> prospective samples were compared to a reference assay's HBeAg
> results. The following table shows this comparison and percent
> agreement with 95% confidence intervals with the reference HBeAg
> assay.

##### Prospective Samples Comparison

  ----------------------------------- ------------ -----------------------
                                      **--**       Reference HBeAg

  Reference Serology Classification   **--**       DiaSorin HBeAg

  Chronic infection                   1            1

  Recovery                            2            2

  Past Infection                      8            8

  HBV vaccine response                38           38

  Not previously infected with HBV    87           87

  Grand Total                         136          136
  ----------------------------------- ------------ -----------------------

Prospective Samples Agreement Rates

  --------------- ----------- --------------------------------------- ------ ----- --------- ------- ---------
  Chronic         Positive                                                   NA              (0/0)   
  Infection       agreement                                                                          
                  with                                                                               
                  reference                                                                          
                  assay                                                                              
                  results =                                                                          

                  95% CI =                                                   NA                      

                              Negative agreement with reference assay 100%         (1/1)             
                              results =                                                              

                              95% CI =                                2.5 to                         
                                                                      100%                           

  Recovery                    Positive agreement with reference assay NA           (0/0)             
                              results =                                                              

                              95% CI =                                NA                             

                              Negative agreement with reference assay 100%                           (2/2)
                              results =                                                              

                              95% CI =                                15.8                           
                                                                      to                             
                                                                      100%                           

  HBV Vaccine                 Positive agreement with reference assay NA           (0/0)             
                              results =                                                              

  Response                    95% CI =                                NA                             

                              Negative agreement with reference assay 100%                           (38/38)
                              results =                                                              

                              95% CI =                                90.8                           
                                                                      to                             
                                                                      100%                           

  Past Infection              Positive agreement with reference assay NA           (0/0)             
                              results =                                                              

                              95% CI =                                NA                             

                              Negative agreement with reference assay 100%         (8/8)             
                              results =                                                              

                              95% CI =                                63.1                           
                                                                      to                             
                                                                      100%                           

  Not Previously              Positive agreement with reference assay NA           (0/0)             
                              results =                                                              

  Infected                    95% CI =                                NA                             

                              Negative agreement with reference assay 100%         (87/87)           
                              results =                                                              

                              95% CI =                                98.5                           
                                                                      to                             
                                                                      100%                           
  --------------- ----------- --------------------------------------- ------ ----- --------- ------- ---------

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-EBK PLUS assay
> in detecting HBeAg. 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 (diagnosed by a
    physician as having HBsAg positive for greater than 6 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.
>
> The study (testing) sites were blinded to the previous specimen
> categorization. All testing was performed by the manual ETI-EBK 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 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-EBK PLUS retrospective clinical
> studies.

  ------------------------------------------------------------------------------------------
  Characterization Based  HBsAg   HBeAg   IgM        Total      Anti-HBe   anti-HBs   n
  On Single Point                         anti-HBc   anti-HBc                         
  Specimen                                                                            
  ----------------------- ------- ------- ---------- ---------- ---------- ---------- ------
  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 \-      \-      \-         \-         \-         \-         343
  with HBV                                                                            

  Uninterpretable         \-      \+ or I \-         \-         \-         \-         13

                          \-      \+      \-         \+         \-         \+         2

                          \-      \+      \-         \+         \+         \+         1

                          \-      I       \-         \+         \-         \-         1
  ------------------------------------------------------------------------------------------

\*I = indeterminate result

Based on the above classifications the ETI-EBK PLUS results for the
retrospective samples were compared to a reference assay's HBeAg results
as determined by the methods above. The following table shows this
comparison and percent agreement with 95% confidence intervals with the
reference HBeAg results.

##### 

##### Retrospective Samples Comparison

  -------------------- ------- ------- ------- ------- ------- ------------------
                       **−**           \+              I^‡^    Reference HBeAg

  Reference Serology   **−**   **+**   **−**   **+**   **−**   DiaSorin HBeAg
  Classification                                               

  Acute infection      5       1       0       54      0       60

  Chronic infection    107     1       2       29      0       139

  Recovery             50      0       0       0       0       50

  HBV vaccine response 20      0       0       0       0       20

  Not previously       343     0       0       0       0       343
  infected with HBV                                            

  Past Infection       20      0       0       0       1       21

  Uninterpretable      0       0       9       1       7       17

  Total                545     2       11      84      8       650
  -------------------- ------- ------- ------- ------- ------- ------------------

> ^‡^ Indeterminate result

Retrospective Samples Agreement Rate

+------------+-----------------------------------------+-----+--------+
| Acute      |                                         |     |        |
| Infection  |                                         |     |        |
+------------+-----------------------------------------+-----+--------+
|            | Positive agreement with reference assay | 1   | (      |
|            | results =                               | 00% | 54/54) |
+------------+-----------------------------------------+-----+--------+
|            | 95% CI =                                | 9   |        |
|            |                                         | 3.4 |        |
|            |                                         | to  |        |
|            |                                         | 1   |        |
|            |                                         | 00% |        |
+------------+-----------------------------------------+-----+--------+
|            | Negative agreement with reference assay | 83  | (5/6)  |
|            | results=                                | .3% |        |
+------------+-----------------------------------------+-----+--------+
|            | 95% CI =                                | 3   |        |
|            |                                         | 5.9 |        |
|            |                                         | to  |        |
|            |                                         | 99  |        |
|            |                                         | .6% |        |
+------------+-----------------------------------------+-----+--------+
| #          |                                         |     |        |
| ## Chronic |                                         |     |        |
|  Infection |                                         |     |        |
+------------+-----------------------------------------+-----+--------+
|            | Positive agreement with reference assay | 93  | (      |
|            | results =                               | .5% | 29/31) |
+------------+-----------------------------------------+-----+--------+
|            | 95% CI =                                | 7   |        |
|            |                                         | 8.6 |        |
|            |                                         | to  |        |
|            |                                         | 99  |        |
|            |                                         | .2% |        |
+------------+-----------------------------------------+-----+--------+
|            | Negative agreement with reference assay | 99  | (10    |
|            | results=                                | .1% | 7/108) |
+------------+-----------------------------------------+-----+--------+
|            | 95% CI =                                | 9   |        |
|            |                                         | 5.0 |        |
|            |                                         | to  |        |
|            |                                         | 1   |        |
|            |                                         | 00% |        |
+------------+-----------------------------------------+-----+--------+
| ##         |                                         |     |        |
| # Recovery |                                         |     |        |
+------------+-----------------------------------------+-----+--------+
|            | Positive agreement with reference assay | NA  | (0/0)  |
|            | results =                               |     |        |
+------------+-----------------------------------------+-----+--------+
|            | 95% CI =                                | NA  |        |
+------------+-----------------------------------------+-----+--------+
|            | Negative agreement with reference assay | 1   | (      |
|            | results=                                | 00% | 50/50) |
+------------+-----------------------------------------+-----+--------+
|            | 95% CI =                                | 9   |        |
|            |                                         | 2.9 |        |
|            |                                         | to  |        |
|            |                                         | 1   |        |
|            |                                         | 00% |        |
+------------+-----------------------------------------+-----+--------+
| ###        |                                         |     |        |
| HBV Vaccin |                                         |     |        |
| e Response |                                         |     |        |
+------------+-----------------------------------------+-----+--------+
|            | Positive agreement with reference assay | NA  | (0/0)  |
|            | results =                               |     |        |
+------------+-----------------------------------------+-----+--------+
|            | 95% CI =                                | NA  |        |
+------------+-----------------------------------------+-----+--------+
|            | Negative agreement with reference assay | 1   | (      |
|            | results=                                | 00% | 20/20) |
+------------+-----------------------------------------+-----+--------+
|            | 95% CI =                                | 8   |        |
|            |                                         | 3.2 |        |
|            |                                         | to  |        |
|            |                                         | 1   |        |
|            |                                         | 00% |        |
+------------+-----------------------------------------+-----+--------+
| ### Not    |                                         |     |        |
|  Previousl |                                         |     |        |
| y Infected |                                         |     |        |
+------------+-----------------------------------------+-----+--------+
|            | Positive agreement with reference assay | NA  | (0/0)  |
|            | results =                               |     |        |
+------------+-----------------------------------------+-----+--------+
|            | 95% CI =                                | NA  |        |
+------------+-----------------------------------------+-----+--------+
|            | Negative agreement with reference assay | 1   | (34    |
|            | results=                                | 00% | 3/343) |
+------------+-----------------------------------------+-----+--------+
|            | 95% CI =                                | 9   |        |
|            |                                         | 8.9 |        |
|            |                                         | to  |        |
|            |                                         | 1   |        |
|            |                                         | 00% |        |
+------------+-----------------------------------------+-----+--------+
| Past       |                                         |     |        |
| Infection  |                                         |     |        |
+------------+-----------------------------------------+-----+--------+
|            | Positive agreement with reference assay | NA  | (0/0)  |
|            | results =                               |     |        |
+------------+-----------------------------------------+-----+--------+
|            | 95% CI =                                | NA  |        |
+------------+-----------------------------------------+-----+--------+
|            | Negative agreement with reference assay | 1   | (      |
|            | results=                                | 00% | 20/20) |
+------------+-----------------------------------------+-----+--------+
|            | 95% CI =                                | 8   |        |
|            |                                         | 3.2 |        |
|            |                                         | to  |        |
|            |                                         | 1   |        |
|            |                                         | 00% |        |
+------------+-----------------------------------------+-----+--------+
| ### Unint  |                                         |     |        |
| erpretable |                                         |     |        |
+------------+-----------------------------------------+-----+--------+
|            | Positive agreement with reference assay | 10  | (1/10) |
|            | results =                               | .0% |        |
+------------+-----------------------------------------+-----+--------+
|            | 95% CI =                                | 0.3 |        |
|            |                                         | to  |        |
|            |                                         | 44  |        |
|            |                                         | .5% |        |
+------------+-----------------------------------------+-----+--------+
|            | Negative agreement with reference assay | NA  | (0/0)  |
|            | results=                                |     |        |
+------------+-----------------------------------------+-----+--------+
|            | 95% CI =                                | NA  |        |
+------------+-----------------------------------------+-----+--------+

11. **Conclusions Drawn from Studies**

> The study data demonstrates that acceptable performance is obtained
> with the DiaSorin ETI-EBK PLUS assay when testing specimens collected
> in serum and plasma. 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-EBK PLUS assay is safe and effective for its stated
> purpose when used as instructed in the package insert. The DiaSorin
> ETI-EBK PLUS HBeAg assay can be stored up to 6 months at 2-8°C

12. **Panel Recommendations**

The Microbiology Advisory Panel met on January 20, 2000, to consider the
safety and effectiveness of the ETI-EBK PLUS assay. The advisory 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 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:

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

```{=html}
<!-- -->
```
-   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 February 5, 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.