**Summary of Safety and Effectiveness**

**IMMULITE and IMMULITE 2000 AFP**

# GENERAL INFORMATION

> Device Generic Name: Enzyme immunometric test system for the
> quantitative measurement of alpha fetoprotein (AFP) in human serum.
>
> Device Trade Name: IMMULITE^^ AFP\*
>
> IMMULITE^^ 2000 AFP\*

Applicant\'s Name and Address: Diagnostic Products Corporation (DPC)

5700 West 96th Street

Los Angeles, California 90045

Premarket Approval Application

(PMA) Number: P010007

Date of Notice of Approval to

the Applicant: November 9, 2001

\* IMMULITE^^ and IMMULITE^^ 2000 are registered trademarks of
Diagnostic Products Corporation. All references herein to IMMULITE or
IMMULITE 2000 are to the registered trademark.

# INDICATIONS FOR USE

> The IMMULITE^^ AFP is intended to be used with the IMMULITE Analyzer
> and IMMULITE 2000^^ AFP is intended to be used with the IMMULITE 2000
> Analyzer. These devices are indicated for *in vitro* diagnostic use
> for the quantitative measurement of alpha-fetoprotein (AFP) in either
> of two contexts: (a) serial measurements in human serum to aid in the
> management of patients with nonseminomatous testicular cancer; or (b)
> measurements in maternal serum and amniotic fluid during gestational
> weeks 15 through 20 -- used in conjunction with ultrasonography or
> amniography -- to aid in detection of fetal open neural tube defects.

# contraindications, 

> No known contraindications.

# WARNING AND PRECAUTIONS 

> See Package Insert for a listing of Warnings and Precautions.

#  {#section .unnumbered}

# DESCRIPTION OF DEVICES

> **Background**
>
> Alpha-fetoprotein (AFP) is a single chain glycoprotein with a
> molecular weight of approximately 70,000 daltons. Discovery of this
> embryo-specific protein in human fetal serum was reported in 1956 by
> Bergstrand and Czar^1^. AFP shares considered sequence homology with
> albumin, but its precise functional role has not been clearly
> defined^2^. AFP appears as a major serum protein in the fetus, reaches
> a peak at 13 weeks of gestation, but its concentration decreases
> rapidly toward birth^3-5^. The reappearance of the elevated AFP
> concentrations in adult serum has been observed during pregnancy and
> in conjunction with several benign liver diseases (hepatitis,
> cirrhosis) and malignancies such as primary hepatocellular carcinoma
> and some germ cell tumors.^6-8^
>
> **Fetal Open Neural Tube Defects**
>
> Since 1972, AFP measurements have become recognized to have clinical
> value as a screening test to aid in the detection of open NTDs^9-13^.
> Elevated levels of AFP in maternal serum and amniotic fluid indicate
> an increased risk for carrying an affected fetus. The prevalence of
> NTDs, estimated to be approximately 1-2 per 1000 live births in the
> United States, varies considerably in different population due to a
> number of factors^13^.

NTDs are a consequence of abnormal development of the central nervous
system during fetal development. The major types of NTDs are classified
as anencephaly and spina bifida (myelomeningocele) which together
constitute about 94% of cases, with the remainder classified as
encephaolocele^14^.

In anencephaly the forebrain fails to develop normally, the bones of the
cranium fail to fuse and the exposed neural tissue degenerates.
Anencephaly lesions are considered open lesions and death usually
results before or shortly after birth. In spina bifida, the neural tube
fails to fuse. Approximately 80% of these lesions are defined as open
with neural tissue exposed or covered only by a thin transparent
membrane. The remainder is defined as closed and covered completely by
skin or by a thick membrane. Closed spina bifida is not amenable to
detection by AFP measurement. Spina bifida results in mild to severe
disability due to impaired innervation of the lower portion of the body
consequent to abnormal development of the spinal column and often the
spinal cord. Finally, encephalocele is the least common of the three
major NTDs and manifests as a closed lesion in approximately 80% of
cases. Most cases of encephalocele are therefore not detectable via AFP
measurement^13^.

[Prenatal Testing for NTDs]{.underline}

During pregnancy, maternal serum alpha-fetoprotein (MSAFP) levels rise
until the mid third trimester as the result of diffusion of fetal AFP
across the placenta into the maternal circulation. Open fetal NTDs are
associated with elevated MSAFP levels during the second trimester of
pregnancy.

When assessing the open NTD risk, maternal factors such as race, weight,
age, diabetes, multiple births and family history have to be taken into
consideration. The optimal time for screening MSAFP is between the
16^th^ and 18^th^ weeks of pregnancy although screening is still
effective before or after this period. Normal median MSAFP levels rise
from approximately 30 to 65 ng/mL during this time frame. The method of
choice in reporting AFP levels is as a Multiple of the Median (MoM).
Values greater than 2.0 to 2.5 MoM are cause for further investigation
for the presence of fetal NTD. Repeating the AFP measurement in a second
sample following an initial elevated MSAFP has proven useful for sorting
out false positive results. However, a repeat MSAFP measurement that
falls within the normal range does not exclude the possibility of an
open fetal malformation, it only reduces the likelihood^13^.

To rule out the more common causes of AFP elevation, the second elevated
serum test is followed by an ultrasound procedure which allows
visualization of multiple pregnancies and enables a more accurate
estimate of gestational age. Certain NTDs, if present, can also be
visualized during an ultrasound procedure. If ultrasonography does not
provide an explanation of an elevated AFP level, the physician may offer
the patient an amniocentesis.

Amniotic fluid APF levels peak at about 12 weeks gestation after which
they rapidly decline and taper off at about week 22 of gestation. A high
level of AFP in the amniotic fluid indicates that a high probability
exists that the fetus has an open NTD, provided other possible
explanations have been ruled out. The high probability of an NTD can be
confirmed by performing an additional acetylcholinesterase test on the
amniotic fluid. Additionally, the location and magnitude of the possible
defects can be assessed by means of either high resolution
ultrasonography or by amniography.

**Nonseminomatous Testicular Cancer Management**

> The quantitative determination of AFP in human serum when used in
> conjunction with other evaluation procedures and diagnostic tests has
> proven to be valuable in the management of patients with
> nonseminomatous testicular cancer. Serial measurements of serum AFP
> following therapy or surgery could monitor effectiveness of the
> therapeutic regimens and disease progression or recurrence.

## IMMULITE AFP

The IMMULITE AFP is a solid-phase, two-site sequential chemiluminescent
immunometric assay. The solid phase, a polystyrene bead enclosed within
an IMMULITE Test Unit, is coated with a monoclonal antibody specific for
AFP.

> The patient sample and a buffer/serum matrix are simultaneously
> introduced into the Test Unit, and incubated for approximately 30
> minutes at 37°C with intermittent agitation. During this time, AFP in
> the sample binds to the monoclonal, anti-AFP antibody-coated bead.
> Unbound sample is then removed by a centrifugal wash.
>
> An alkaline phosphatase-labeled polyclonal anti-AFP antibody is
> introduced, and the Test Unit is incubated for another 30-minute
> cycle. The unbound enzyme conjugate is removed by a centrifugal wash.
> Substrate is then added, and the Test Unit is incubated for a further
> 10 minutes.
>
> The chemiluminescent substrate, a phosphate ester of adamantyl
> dioxetane, undergoes hydrolysis in the presence of alkaline
> phosphatase to yield an unstable intermediate. The continuous
> production of this intermediate results in the sustained emission of
> light, providing a window for multiple readings. The bound complex -
> and thus the photon output, as measured by the luminometer - is
> proportional to the concentration of AFP in the sample. The
> concentration of AFP in the patient sample is obtained using a stored
> master calibration curve within the IMMULITE analyzer.

## IMMULITE 2000 AFP

IMMULITE 2000 AFP is a solid-phase, two-site chemiluminescent
immunometric assay. The solid phase is a polystyrene bead coated with a
monoclonal antibody specific for AFP.

> The patient serum sample and a buffer/serum matrix are introduced into
> the Reaction Tube containing the bead and incubated for approximately
> 30 minutes at 37°C with agitation. During this time, AFP in the sample
> binds to the monoclonal, anti-AFP antibody-coated bead. Unbound sample
> is then removed by a centrifugal wash.
>
> An alkaline phosphatase-labeled polyclonal anti-AFP antibody is
> introduced and the Reaction Tube is incubated for another 30 minute
> cycle. The unbound enzyme conjugate is removed by a centrifugal wash.
> Substrate is then added, and the Reaction Tube is incubated for a
> further 5 minutes. The concentration of AFP in the sample is then
> measured in the same manner as that of IMMULITE AFP.

# ALTERNATIVE PRACTICES OR PROCEDURES

The detection of NTDs in fetuses can be achieved by using the following
alternative practices or procedures:

1\) Ultrasonography;

2\) Amniography;

3\) Amniotic fluid acetylcholinesterase testing;

4\) The use of other legally-marketed AFP devices.

In the management of nonseminomatous testicular cancer, alternative and
additional practices include physical evaluation, histological diagnosis
after orchiectomy, lymphadenectomy and lymph node biopsy, exploratory
laparoscopy, lymphography, chest radiography, ultrasound, computed
tomography, magnetic resonance imaging, and other immunological devices
for the quantification of other markers in human serum.

# MARKETING HISTORY

IMMULITE AFP and IMMULITE 2000 AFP have been marketed in the United
States and Internationally as an aid in the management of patients with
nonseminomatous testicular cancer since December 1998.

# POTENTIAL ADVERSE EFFECTS OF DEVICE ON HEALTH

The extensive follow-up testing to maternal serum and amniotic fluid AFP
measurement reduces to a very low likelihood, but does not completely
eliminate the possibility that a healthy fetus may be incorrectly
diagnosed as having an NTD. Special consideration needs to be given when
interpreting AFP results from pregnancies where one twin appears by
ultrasound to have suffered fetal demise or an open NTD while the other
appears unaffected in order to avoid a misdiagnosis of the unaffected
twin. In addition, an amniocentesis procedure that may be performed as a
follow-up to an elevated MSAFP result carries a miscarriage risk,
estimated to be approximately 1 in 1000. The physician should discuss
the risk of miscarriage with the patient.

Patients undergoing treatment for nonseminomatous testicular cancer
should not experience any adverse effects if test results from these in
vitro devices are used as an aid in managing the cancer in conjunction
with other routine medical practices and procedures and all available
clinical information. A false positive result would indicate that a
person may be incorrectly diagnosed as having testicular cancer whereas
a false negative result would indicate no change in the patient's
clinical status.

# SUMMARY OF STUDIES

## Nonclinical Studies

The information in the table below provides the performance
characteristics of IMMULITE AFP and IMMULITE 2000 AFP.

+----------------------+-----------------------+-----------------------+
| **Characteristics**  | **IMMULITE AFP**      | **IMMULITE 2000 AFP** |
+======================+=======================+=======================+
| Calibration upper    | 300 IU/mL             | 300 IU/mL             |
| limit                |                       |                       |
+----------------------+-----------------------+-----------------------+
| Analytical           | 0.2 IU/mL             | 0.2 IU/mL             |
| Sensitivity          |                       |                       |
+----------------------+-----------------------+-----------------------+
| Precision            | CV range: 4.5-7.4%    | CV range: 2.1-6.3%    |
| (within-run)         |                       |                       |
+----------------------+-----------------------+-----------------------+
| Precision (total)    | CV range: 5.8-8.4%    | CV range: 4.5-12%     |
+----------------------+-----------------------+-----------------------+
| Specificity          | No clinically         | No clinically         |
|                      | significant           | significant           |
|                      |                       |                       |
|                      | cross-reactivity      | cross-reactivity      |
+----------------------+-----------------------+-----------------------+
| Linearity            | %Observed/Expected    | %Observed/Expected    |
|                      | range:                | range:                |
| (Serum)              |                       |                       |
|                      | 97-114%               | 96-111%               |
+----------------------+-----------------------+-----------------------+
| Linearity            | %Observed/Expected    | %Observed/Expected    |
|                      | range:                | range:                |
| (Amniotic Fluid)     |                       |                       |
|                      | 81-119%               | 94-106%               |
+----------------------+-----------------------+-----------------------+
| Spiking Recovery     | %Observed/Expected    | %Observed/Expected    |
|                      | range:                | range:                |
| (Serum)              |                       |                       |
|                      | 98-108%               | 94-106%               |
+----------------------+-----------------------+-----------------------+
| Spiking Recovery     | %Observed/Expected    | %Observed/Expected    |
|                      | range:                | range:                |
| (Amniotic)           |                       |                       |
|                      | 81-127%               | 96-128%               |
+----------------------+-----------------------+-----------------------+
| Effect of Bilirubin  | Small but             | Small but             |
|                      | statistically         | statistically         |
| (unconjugated)       | significant effect    | significant effect    |
+----------------------+-----------------------+-----------------------+
| Effect of Lipemia    | No significant effect | No significant effect |
+----------------------+-----------------------+-----------------------+
| Effect of Hemolysis  | No significant effect | No significant effect |
+----------------------+-----------------------+-----------------------+
| No \"high dose       | 450,000 IU/mL         | 534,000 IU/mL         |
| hook\" effect        |                       |                       |
|                      |                       |                       |
| up to AFP levels of: |                       |                       |
+----------------------+-----------------------+-----------------------+
| Stability            | Kit is stable for at  | Kit is stable for at  |
|                      | least one year when   | least one year when   |
|                      | kept in long-term     | kept in long-term     |
|                      | storage conditions.   | storage conditions.   |
+----------------------+-----------------------+-----------------------+

## Clinical studies

### Expected Values

> Clinical studies were performed at three medical institutions
> involving 2373 maternal serum and 385 amniotic fluid samples
> prospectively and retrospectively obtained during 15 through 20 weeks
> of gestation. These samples were assayed by IMMULITE and IMMULITE 2000
> AFP.
>
> Of the samples tested, 2287 maternal sera and 341 amniotic fluid
> specimens were confirmed as unaffected, non-insulin-dependent,
> singleton pregnancies at 15 through 20 gestational weeks. The
> following table shows the medians and multiples of medians calculated
> by the weighted log-linear regression for the 2287 serum samples,
> tested either by IMMULITE or IMMULITE 2000 AFP.
>
> ![](media/image1.wmf){width="4.729861111111111in" height="1.4in"}
>
> The following table shows the medians and multiples of medians
> calculated by the weighted log-linear regression for these 341
> amniotic fluid samples, tested either by IMMULITE or IMMULITE 2000
> AFP.
>
> ![](media/image2.wmf){width="4.7in" height="1.3645833333333333in"}

### Clinical Specificity and Sensitivity

> Using the above cutoffs, the clinical specificity and clinical
> sensitivity were calculated to evaluate the ability of the IMMULITE
> and IMMULITE 2000 AFP to confirm unaffected pregnancies (clinical
> specificity) and to detect open NTD pregnancies (clinical
> sensitivity).
>
> ![](media/image3.wmf){width="4.405555555555556in"
> height="2.0902777777777777in"}
>
> ![](media/image4.wmf){width="4.342361111111111in"
> height="2.0902777777777777in"}
>
> ![](media/image5.wmf){width="4.405555555555556in"
> height="1.0520833333333333in"}
>
> ![](media/image6.wmf){width="4.198611111111111in"
> height="1.0520833333333333in"}
>
> ![](media/image7.wmf){width="4.659027777777778in"
> height="2.0902777777777777in"}
>
> ![](media/image8.wmf){width="4.736111111111111in"
> height="2.0902777777777777in"}
>
> ![](media/image9.wmf){width="4.659027777777778in"
> height="1.0520833333333333in"}
>
> ![](media/image10.wmf){width="4.736111111111111in"
> height="1.0520833333333333in"}

###  {#section-1 .unnumbered}

###  {#section-2 .unnumbered}

###  {#section-3 .unnumbered}

###  {#section-4 .unnumbered}

###  {#section-5 .unnumbered}

###  {#section-6 .unnumbered}

###  {#section-7 .unnumbered}

### Conclusions

> In previously conducted studies under the 510(k)s (K983263 & K983263),
> the IMMULITE and IMMULITE 2000 AFP were cleared for serial
> measurements in human serum to aid in the management of patients with
> nonseminomatous testicular cancer and other conditions associated with
> abnormal AFP production. This indication has been further expanded
> based upon the additional clinical performance studies to include AFP
> measurements in maternal serum and amniotic fluid during gestational
> weeks 15 through 20 -- used in conjunction with ultrasonography or
> amniography -- to aid in detection of fetal open neural tube defects.

## Method Comparison Studies

### IMMULITE AFP

#### Nonseminomatous testicular cancer management

> The assay was compared to a legally marketed AFP assay (Kit C), at two
> clinical sites. At the first (in the northwestern United States), a
> total of 264 specimens were evaluated, including samples from male
> patients with nonseminomatous testicular cancer and other malignant
> and nonmalignant conditions, as well as samples from a few female
> patients. The results were compared qualitatively, relative to cutoffs
> based on the stated 99th percentiles for healthy males --- namely, 5
> IU/mL for IMMULITE, and 8.9 ng/mL (7.36 IU/mL) for another legally
> marketed device.

Relative Relative

IMMULITE AFP Sensitivity Specificity

[Kit C Pos Neg (95% CI) (95% CI)]{.underline}

Pos 74 4 94.9% 97.3%

[Neg 5 181 (87.4-98.6%) (93.8-99.1%)]{.underline}

Agreement: 96.6%

> At the second site (in the southern United States), a total of 213
> specimens were studied, including samples from male patients with
> seminomatous and nonseminomatous testicular cancer, and other
> malignant conditions, as well as samples from a few female patients.

Relative Relative

IMMULITE AFP Sensitivity Specificity

[Kit C Pos Neg (95% CI) (95% CI)]{.underline}

Pos 62 3 95.4% 97.3%

[Neg 4 144 (87.1-99.0%) (93.2-99.3%)]{.underline}

Agreement: 96.7%

> The 424 results from the two sites which were within range by both
> assays were also compared by linear regression analysis:

(IML) = 0.83 (Kit C) -- 0.17 IU/mL r = 0.99

95% CI Slope Intercept

Lower 0.81 -0.65

Upper 0.84 0.31

#### Open Neural Tube Defects

##### Maternal serum

> In two separate clinical studies conducted in the United States,
> IMMULITE AFP results were compared to two legally marketed AFP assays
> (Kit A and Kit B) in a linear regression for maternal serum samples in
> the range from nondetectable to 300 IU/mL.

(IML) = 0.83 (Kit B) + 6.51 IU/mL r = 0.97 n = 1006

95% CI Slope Intercept

Lower 0.82 5.93

Upper 0.85 7.09

(IML) = 0.87 (Kit A) + 2.72 IU/mL r = 0.96 n = 346

95% CI Slope Intercept

Lower 0.84 1.64

Upper 0.89 3.79

##### Amniotic fluid

> In one of the studies above, IMMULITE AFP results were also compared
> to a Kit B in a linear regression for amniotic fluid samples in the
> range from nondetectable to 173 kIU/mL\*.

(IML) = 0.78 (Kit B) + 0.17 kIU/mL r = 0.99 n = 185

95% CI Slope Intercept

Lower 0.76 -0.19

Upper 0.79 0.53

\* Amniotic fluid samples were diluted 1-in-101 off-line before they
were tested by the IMMULITE instrument.

### IMMULITE 2000 AFP

#### Nonseminomatous testicular cancer management

> The IMMULITE 2000 AFP assay was compared to IMMULITE AFP on a total of
> 205 samples from male patients in different clinical stages, pre- and
> post-surgery, of nonseminomatous testicular cancer. (Concentration
> range: approximately 0.3 to 280 IU/mL.) By linear regression:

(IML 2000) = 1.04 (IML) + 0.34 IU/mL r = 0.998 n = 205

95% CI Slope Intercept

Lower 1.03 -0.51

Upper 1.05 1.20

#### Open NTD

##### Maternal serum

> In two separate clinical studies conducted in the United States,
> IMMULITE 2000 AFP results were compared to two legally marketed assays
> (Kit A and Kit B) in a linear regression for maternal serum samples in
> the range from nondetectable to 300 IU/mL.

(IML 2000) = 0.91 (Kit A) + 1.81 IU/mL r = 0.98 n = 346

95% CI Slope Intercept

Lower 0.89 0.94

Upper 0.93 2.68

(IML 2000) = 0.73 (Kit B) + 5.22 IU/mL r = 0.97 n = 1015

95% CI Slope Intercept

Lower 0.72 4.66

Upper 0.74 5.79

##### Amniotic fluid

> In one of the studies above, IMMULITE 2000 AFP results were compared
> to Kit B in a linear regression for amniotic fluid samples in the
> range from nondetectable to 286 kIU/mL\*.

(IML 2000) = 0.79 (Kit B) + 2.27 kIU/mL r = 0.99 n = 200

95% CI Slope Intercept

Lower 0.77 1.78

Upper 0.81 2.76

#### Comparison of IMMULITE AFP and IMMULITE 2000 AFP

##### Amniotic fluid

> IMMULITE 2000 AFP was also compared to IMMULITE AFP on 46 amniotic
> fluid samples, in a range from approximately 3 to 20 kIU/mL\*. By
> linear regression:

(IML 2000) = 1.03 (IML) + 0. 52 kIU/mL r = 0.982 n = 46

95% CI Slope Intercept

Lower 0.93 -0.50

Upper 1.12 1.54

Mean of IMMULITE AFP = 10.0 kIU/mL

Mean of IMMULITE 2000 AFP = 10.8 kIU/mL

\* Amniotic fluid samples were diluted 1-in-101 automatically by the
IMMULITE 2000 instrument.

##### Maternal serum

> IMMULITE 2000 AFP was also compared to IMMULITE AFP on 346 maternal
> serum samples in a range from approximately 10 to 120 IU/mL. By linear
> regression:

IMMULITE 2000 = 1.01 × IMMULITE + 0.154 (IU/mL) r = 0.982 n = 346

95% CI Slope Intercept

Lower 0.99 -0.60

Upper 1.03 0.91

Mean of IMMULITE AFP = 33.8 IU/mL

Mean of IMMULITE 2000 AFP = 34.3 IU/mL

# CONCLUSION

# A false positive result could result in the possibility that a healthy fetus may be incorrectly diagnosed as having an NTD. However, the follow-up testing to maternal serum and amniotic fluid AFP measurement reduces the risk to a very low likelihood. Therefore, the benefits of AFP testing far outweigh the risk. {#a-false-positive-result-could-result-in-the-possibility-that-a-healthy-fetus-may-be-incorrectly-diagnosed-as-having-an-ntd.-however-the-follow-up-testing-to-maternal-serum-and-amniotic-fluid-afp-measurement-reduces-the-risk-to-a-very-low-likelihood.-therefore-the-benefits-of-afp-testing-far-outweigh-the-risk. .unnumbered}

The data presented in the submission provide reasonable assurance that
the IMMULITE AFP and IMMULITE 2000 devices are safe and effective as an
aid in the detection of open neural tube defects and as an aid in the
management of nonseminomatous testicular cancer patients.

# PANEL RECOMMENDATIONS

> Pursuant to Section 515(c)(2) of the act as amended by the Safe
> Medical Devices Act of 1990, this PMA was not the subject of an FDA
> Immunology Devices Advisory Panel meeting because the information in
> the PMA substantially duplicated information previously reviewed by
> this Panel.

#  {#section-8 .unnumbered}

# CDRH DECISION

The applicant\'s manufacturing facilities were inspected in October 4,
2001, and the facilities were found to be in compliance with the Good
Manufacturing Practice (GMP) Regulations.

FDA issued an approval order on November 9, 2001.

# APPROVAL SPECIFICATION

Directions for use: See the labeling.

Hazards to Health from Use of the Device: See Indications,
Contraindications, Warnings, precautions and Adverse Events in the
labeling.

Postapproval Requirements and Restrictions: See approval order

# REFERENCES

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3.  E. Ruoslahti and M. Seppala, Studies of carcino-fetal proteins:
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9.  D.J. Brock and R.G. Sutcliffe, Alpha-fetoprotein in the antenatal
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10. A. Milunsky and E. Alpert, The value of alpha-fetoprotein in
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11. D.J.H. Brock, A.E. Bolton, and J.M. Monaghan, Prenatal diagnosis of
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12. A.D. Smith, N.J. Wald, H.S. Cuckle, et al., Amniotic-fluid
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13. J.E. Haddow, et al., Fetal disorders associated with elevated MSAFP
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14. N. Wald, H. Cuckle, and K. Nanchahal, Amniotic fluid
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