Summary of Safety and Effectiveness Data

1.  General Information

> 1.1. Device Generic Name: In vitro nucleic-acid amplification test for
> qualitatively detecting *Hepatitis C virus* (HCV) RNA in human serum
> or plasma from blood collected in EDTA

1.2. Device Trade Name: [COBAS]{.smallcaps} AMPLICOR^TM^ [HCV
T]{.smallcaps}est[,]{.smallcaps} v2.0

1.3. Applicant's Name and Address:

Roche Molecular Systems, Inc. (RMS)

4300 Hacienda Drive

> Pleasanton, CA 94588

1.4. Premarket Approval Application (PMA) Number: P000012

1.5. Date of Panel Recommendation: July 28, 2000

1.6. Date of Notice of Approval : July 3, 2001

2\. Indications for Use

> The COBAS AMPLICOR HCV Test, v2.0 is an *in vitro* diagnostic,
> nucleic-acid amplification test for qualitative detection of HCV RNA
> in human serum or plasma from blood collected in EDTA. This test
> detects by reverse-transcribing target HCV RNA into cDNA, amplifying
> cDNA by polymerase chain reaction (PCR), hybridizing amplified cDNA
> with an oligonucleotide probe that binds enzyme, and catalyzing
> conversion of substrate to a colored product that is recognized by the
> COBAS AMPLICOR Analyzer. The COBAS AMPLICOR HCV Test, v2.0 is
> indicated for patients who have evidence of liver disease and antibody
> evidence of HCV infection**,** and who are suspected to be actively
> infected with HCV. Detection of HCV RNA indicates that the virus is
> replicating and therefore is evidence of active HCV infection.

3\. Contraindications

> None known.

**4. Warnings and Precautions**

> The COBAS AMPLICOR HCV Test, v2.0 is for *in vitro* diagnostic use
> only. Refer to labeling for a listing to other warnings and
> precautions.

**5. Device Description**

5.1. Test Principles

The COBAS AMPLICOR HCV Test, v2.0 is based on five major processes:

-   specimen preparation

-   reverse transcription to generate cDNA from target HCV RNA and HCV
    Internal Control (HCV IC) RNA

-   PCR amplification of target cDNAs by using HCV-specific primers

-   hybridization of amplified cDNAs to target-specific oligonucleotide
    probes

-   colorimetric detection of the probe-bound amplified cDNAs.

> The COBAS AMPLICOR HCV Test, v2.0 permits simultaneous reverse
> transcription and PCR amplification of HCV and HCV IC target RNAs. The
> Master Mix reagent contains a primer pair that is specific for both
> HCV and HCV IC RNAs. Detection of amplified DNA is performed using
> target-specific oligonucleotide probes that permit independent
> identification of HCV amplicon and HCV IC amplicon.

5.1.1. Specimen Preparation

> HCV RNA is isolated directly from serum or EDTA plasma by lysing virus
> particles with a chaotropic agent. HCV IC RNA, introduced into each
> specimen with Lysis Reagent, serves as an extraction and amplification
> control for each processed specimen. HCV and HCV IC RNAs are
> precipitated by using alcohol and then resuspended in Specimen
> Diluent.

5.1.2. Enzyme and Target Selection

> ***Enzyme***: Reactions are catalyzed by thermostable, recombinant
> *Thermus thermophilus* DNA Polymerase (r*Tth* pol). In the presence of
> manganese ion (Mn^2+^) and the appropriate buffer, r*Tth* pol has
> reverse transcriptase and DNA polymerase activities. This allows both
> reverse transcription and PCR amplification to occur in the same
> reaction mixture.
>
> ***Target selection***: Selection of primers and probe was critical
> for COBAS AMPLICOR HCV Test, v2.0 detection of all recognized
> genotypes (see Non-Clinical Performance section of the Package
> Insert). Accordingly, selection of the target RNA sequence was based
> on identifying an HCV-genome region that was maximally conserved among
> genotypes. HCV RNA sequences are most conserved in the 5\'
> untranslated region (UTR). The COBAS AMPLICOR HCV Test, v2.0 uses
> primers KY78 and KY80 to amplify a 5\' UTR sequence of 244
> nucleotides. HCV RNA sequence corresponding to these primers and the
> capture probe are located in the most conserved 5\' UTR domains.

5.1.3. Reverse Transcription

> Processed specimens are added to the amplification mixture in
> amplification tubes (A-tubes) in which reverse transcription and PCR
> amplification occur. The downstream or antisense primer (KY78) is
> biotinylated at its 5\' end; the upstream or sense primer (KY80) is
> not biotinylated. The reaction mixture is heated in the COBAS AMPLICOR
> Analyzer to allow specific annealing of the downstream primer to
> target HCV and HCV IC RNAs. In the presence of Mn^2+^ and excess
> deoxynucleoside triphosphates (dNTPs), including deoxyadenosine,
> deoxyguanosine, deoxycytidine and deoxyuridine (in place of thymidine)
> triphosphates, r*Tth* pol extends the annealed primer to form cDNA.

5.1.4. Target Amplification

> Following reverse transcription of target HCV and HCV IC RNAs, the
> reaction mixture is heated to denature RNA:cDNA hybrids and expose
> sequences that anneal with the primers. As the mixture cools, the
> upstream primer (KY80) anneals specifically to the cDNA strand
> representing each target RNA, r*Tth* pol extends the primer, and a
> second DNA strand is synthesized. This completes the first cycle of
> PCR, yielding a double-stranded DNA copy of each RNA target region
> (HCV and HCV IC).
>
> The reaction mixture is heated again to separate the double-stranded
> DNA and expose the primer-annealing sequences. As the mixture cools,
> primers KY78 and KY80 anneal to target DNA. The r*Tth* pol enzyme, in
> the presence of Mn^2+^ and excess dNTPs, extends the annealed primers
> along the target templates to produce a 244 base pair double-stranded
> DNA \"amplicon.\" The COBAS AMPLICOR Analyzer automatically repeats
> this process for 37 cycles, with each cycle intended to double the
> amount of amplicon DNA. The required number of cycles is preprogrammed
> into the COBAS AMPLICOR Analyzer. Amplification occurs only in the
> region of the HCV genome between the primers; the entire genome is not
> amplified.

5.1.4.1. Amplification of HCV Internal Control (HCV IC) cDNA

> In enzyme-based amplification processes such as PCR, inhibitors that
> may be present in the clinical specimen can reduce amplification
> efficiency. The HCV IC has been added to the COBAS AMPLICOR HCV Test,
> v2.0 to permit identification of processed specimens containing
> substances that may interfere with PCR amplification or samples in
> which HCV RNA may have been lost during sample processing. The HCV IC
> is an RNA transcript with primer-annealing regions identical to those
> in the HCV genome, a randomized internal sequence of similar length
> and base composition as the HCV target sequence, and a unique
> probe-binding region that differentiates HCV IC amplicon from HCV
> amplicon. These features were selected to ensure equivalent
> amplification of HCV IC and HCV target RNAs. The HCV IC is introduced
> into each specimen with the Lysis Reagent and serves as an extraction
> and amplification control for each processed specimen.

5.1.4.2. Selective Amplification

> Selective amplification of target nucleic acid from the clinical
> specimen is achieved in the COBAS AMPLICOR HCV Test, v2.0 by the use
> of AmpErase^®^ (uracil-N-glycosylase) and deoxyuridine triphosphate
> (dUTP). AmpErase recognizes and catalyzes the destruction of DNA
> strands containing deoxyuridine, but not DNA containing thymidine.
> Deoxyuridine is not present in naturally occurring DNA, but is always
> present in amplicon from this assay due to the use of deoxyuridine
> triphosphate in place of thymidine triphosphate as one of the dNTPs in
> the Master Mix reagent; therefore, only amplicon contains
> deoxyuridine.
>
> Deoxyuridine renders contaminating amplicon susceptible to destruction
> by AmpErase prior to amplification of the target DNA. AmpErase, which
> is included in the Master Mix reagent, catalyzes cleavage of
> deoxyuridine-containing DNA at deoxyuridine residues by opening the
> deoxyribose chain at the C1-position. When heated in the first thermal
> cycling step at the alkaline pH of Master Mix, the amplicon DNA chain
> breaks at the position of the deoxyuridine, thereby rendering the DNA
> non-amplifiable. AmpErase is inactive at temperatures above 55°C
> (i.e., throughout the thermal cycling steps) and therefore does not
> destroy target amplicon. Following amplification, any residual
> AmpErase is denatured by the addition of Denaturation Solution,
> thereby preventing the degradation of any target amplicon. AmpErase in
> the COBAS AMPLICOR HCV Test, v2.0 has been demonstrated to inactivate
> at least 10^3^ copies of deoxyuridine-containing HCV amplicon per PCR.

5.1.5. Hybridization Reaction

> Following PCR amplification, the COBAS AMPLICOR Analyzer automatically
> adds Denaturation Solution to the A-tubes to chemically denature the
> HCV amplicon and the HCV IC amplicon to form single-stranded DNA.
> Aliquots of denatured amplicon are then transferred to detection cups
> (D-cups). A suspension of magnetic particles coated with an
> oligonucleotide probe specific for HCV (KY150) or HCV IC (SK535) is
> added to the individual D-cups. The biotin-labeled HCV and HCV IC
> amplicon are hybridized to the target-specific oligonucleotide probes
> bound to the magnetic particles. This hybridization of amplicon to the
> target-specific probe increases the overall specificity of the test.

5.1.6. Detection Reaction

> Following the hybridization reaction, the COBAS AMPLICOR Analyzer
> washes the magnetic particles in the D-cup to remove unbound material
> and then adds Avidin-Horseradish Peroxidase Conjugate. The
> Avidin-Horseradish Peroxidase Conjugate binds to the biotin-labeled
> amplicon that was hybridized to target-specific oligonucleotide probes
> (HCV or HCV IC) bound to the magnetic particles. The COBAS AMPLICOR
> Analyzer removes unbound conjugate by washing the magnetic particles
> and then adds a substrate solution containing hydrogen peroxide and
> 3,3\',5,5\'-tetramethylbenzidine (TMB) to each D-cup. In the presence
> of hydrogen peroxide, particle-bound horseradish peroxidase catalyzes
> the oxidation of TMB to form a colored complex, the absorbance of
> which is measured by the COBAS AMPLICOR Analyzer at 660 nm (A~660~).

5.2. Kit Configuration and Components

> The COBAS AMPLICOR HCV Test, v2.0 consists of eight separately
> packaged kits containing the controls and reagents required for
> specimen preparation, reverse transcription, and PCR amplification and
> detection of HCV and Internal Control target RNA (Table 1).

1.  COBAS AMPLICOR HCV Test, v2.0 Kits

+---------------------------------------------+------+----------------+
| AMPLICOR HCV Specimen Preparation Kit,      | 96   | P/N: 21111086  |
| version 2.0                                 | T    |                |
|                                             | ests | US: 83126      |
+---------------------------------------------+------+----------------+
| AMPLICOR HCV Controls Kit, version 2.0      | 8    | P/N: 21111175  |
|                                             | Sets |                |
|                                             |      | US: 83131      |
+---------------------------------------------+------+----------------+
| AMPLICOR HCV Amplification Kit, version 2.0 | 96   | P/N: 21111094  |
|                                             | T    |                |
|                                             | ests | US: 83127      |
+---------------------------------------------+------+----------------+
| COBAS AMPLICOR HCV Detection Kit, version   | 100  | P/N: 21111132  |
| 2.0                                         | T    |                |
|                                             | ests | US: 83130      |
+---------------------------------------------+------+----------------+
| COBAS AMPLICOR Detection Reagents Kit       | 100  | P/N: 20757470  |
|                                             | T    |                |
|                                             | ests | US: 83276      |
+---------------------------------------------+------+----------------+
| COBAS AMPLICOR Conjugate Detection Reagent  | 200  | P/N: 20764213  |
|                                             | T    |                |
|                                             | ests | US: 83305      |
+---------------------------------------------+------+----------------+
| COBAS AMPLICOR Wash Buffer                  | 500  | P/N: 20759899  |
|                                             | T    |                |
|                                             | ests | US: 83314      |
+---------------------------------------------+------+----------------+
| COBAS AMPLICOR Internal Control Detection   | 100  | P/N: 20757608  |
| Kit                                         | T    |                |
|                                             | ests | US: 83281      |
+---------------------------------------------+------+----------------+

The components of each kit are listed below (Table 2).

2.  Components of the COBAS AMPLICOR HCV Test, v2.0

+--------------------+------+-----------------------------------------+
| *KIT COMPONENT*    |      | ### *FORMULATION*                       |
+--------------------+------+-----------------------------------------+
| **AMPLICOR HCV     |      |                                         |
| Specimen           |      |                                         |
| Preparation Kit,   |      |                                         |
| version 2.0**      |      |                                         |
+--------------------+------+-----------------------------------------+
| (1) HCV Lysis      | Tris |                                         |
|     Reagent, v2.0  | -HCl |                                         |
|                    | bu   |                                         |
| > **HCV LYS,       | ffer |                                         |
| > v2.0**           | co   |                                         |
|                    | ntai |                                         |
|                    | ning |                                         |
|                    | 68%  |                                         |
|                    | g    |                                         |
|                    | uani |                                         |
|                    | dine |                                         |
|                    | thio |                                         |
|                    | cyan |                                         |
|                    | ate, |                                         |
|                    | 3%   |                                         |
|                    | di   |                                         |
|                    | thio |                                         |
|                    | thre |                                         |
|                    | itol |                                         |
|                    | and  |                                         |
|                    | \<1% |                                         |
|                    | g    |                                         |
|                    | lyco |                                         |
|                    | gen, |                                         |
|                    | 8 x  |                                         |
|                    | 6.9  |                                         |
|                    | mL   |                                         |
+--------------------+------+-----------------------------------------+
| (2) HCV Specimen   | Tris |                                         |
|     Diluent, v2.0  | -HCl |                                         |
|                    | bu   |                                         |
| > **HCV DIL,       | ffer |                                         |
| > v2.0**           | con  |                                         |
|                    | tain |                                         |
|                    | ing, |                                         |
|                    | \<0. |                                         |
|                    | 005% |                                         |
|                    | poly |                                         |
|                    | rA   |                                         |
|                    | RNA  |                                         |
|                    | (syn |                                         |
|                    | thet |                                         |
|                    | ic), |                                         |
|                    | EDTA |                                         |
|                    | and  |                                         |
|                    | 0    |                                         |
|                    | .05% |                                         |
|                    | so   |                                         |
|                    | dium |                                         |
|                    | az   |                                         |
|                    | ide, |                                         |
|                    | 8 x  |                                         |
|                    | 4.8  |                                         |
|                    | mL   |                                         |
+--------------------+------+-----------------------------------------+
| (3) HCV Internal   | A    |                                         |
|     Control, v2.0  | solu |                                         |
|                    | tion |                                         |
| > **HCV IC, v2.0** | co   |                                         |
|                    | ntai |                                         |
|                    | ning |                                         |
|                    | \<0. |                                         |
|                    | 001% |                                         |
|                    | no   |                                         |
|                    | n-in |                                         |
|                    | fect |                                         |
|                    | ious |                                         |
|                    | *in  |                                         |
|                    | vi   |                                         |
|                    | tro* |                                         |
|                    | tra  |                                         |
|                    | nscr |                                         |
|                    | ibed |                                         |
|                    | RNA  |                                         |
|                    | (mi  |                                         |
|                    | crob |                                         |
|                    | ial) |                                         |
|                    | enco |                                         |
|                    | ding |                                         |
|                    | HCV  |                                         |
|                    | pr   |                                         |
|                    | imer |                                         |
|                    | bin  |                                         |
|                    | ding |                                         |
|                    | s    |                                         |
|                    | eque |                                         |
|                    | nces |                                         |
|                    | and  |                                         |
|                    | a    |                                         |
|                    | un   |                                         |
|                    | ique |                                         |
|                    | p    |                                         |
|                    | robe |                                         |
|                    | bin  |                                         |
|                    | ding |                                         |
|                    | reg  |                                         |
|                    | ion, |                                         |
|                    | \<0. |                                         |
|                    | 005% |                                         |
|                    | poly |                                         |
|                    | rA   |                                         |
|                    | RNA  |                                         |
|                    | (syn |                                         |
|                    | thet |                                         |
|                    | ic), |                                         |
|                    | E    |                                         |
|                    | DTA, |                                         |
|                    | and  |                                         |
|                    | 0    |                                         |
|                    | .05% |                                         |
|                    | so   |                                         |
|                    | dium |                                         |
|                    | az   |                                         |
|                    | ide, |                                         |
|                    | 8 x  |                                         |
|                    | 0.1  |                                         |
|                    | mL   |                                         |
+--------------------+------+-----------------------------------------+
| **AMPLICOR HCV     |      |                                         |
| Controls Kit,      |      |                                         |
| version 2.0**      |      |                                         |
+--------------------+------+-----------------------------------------+
| (4) Negative       | H    |                                         |
|     Plasma (Human) | uman |                                         |
|                    | pl   |                                         |
| > **NHP**          | asma |                                         |
|                    | non- |                                         |
|                    | reac |                                         |
|                    | tive |                                         |
|                    | by   |                                         |
|                    | FDA  |                                         |
|                    | lice |                                         |
|                    | nsed |                                         |
|                    | t    |                                         |
|                    | ests |                                         |
|                    | for  |                                         |
|                    | anti |                                         |
|                    | body |                                         |
|                    | to   |                                         |
|                    | HCV, |                                         |
|                    | anti |                                         |
|                    | body |                                         |
|                    | to   |                                         |
|                    | H    |                                         |
|                    | IV-1 |                                         |
|                    | and  |                                         |
|                    | HI   |                                         |
|                    | V-2, |                                         |
|                    | HIV  |                                         |
|                    | p24  |                                         |
|                    | ant  |                                         |
|                    | igen |                                         |
|                    | and  |                                         |
|                    | HB   |                                         |
|                    | sAg, |                                         |
|                    | and  |                                         |
|                    | co   |                                         |
|                    | ntai |                                         |
|                    | ning |                                         |
|                    | 0.1% |                                         |
|                    | Pr   |                                         |
|                    | oCli |                                         |
|                    | n^^ |                                         |
|                    | 300, |                                         |
|                    | 8 x  |                                         |
|                    | 0.6  |                                         |
|                    | mL   |                                         |
+--------------------+------+-----------------------------------------+
| (5) HCV (-)        | A    |                                         |
|     Control, v2.0  | solu |                                         |
|                    | tion |                                         |
| > **HCV (-) C,     | co   |                                         |
| > v2.0**           | ntai |                                         |
|                    | ning |                                         |
|                    | \<0. |                                         |
|                    | 005% |                                         |
|                    | poly |                                         |
|                    | rA   |                                         |
|                    | RNA  |                                         |
|                    | (syn |                                         |
|                    | thet |                                         |
|                    | ic), |                                         |
|                    | EDTA |                                         |
|                    | and  |                                         |
|                    | 0    |                                         |
|                    | .05% |                                         |
|                    | so   |                                         |
|                    | dium |                                         |
|                    | az   |                                         |
|                    | ide, |                                         |
|                    | 8 x  |                                         |
|                    | 0.1  |                                         |
|                    | mL   |                                         |
+--------------------+------+-----------------------------------------+
| (6) HCV (+)        | A    |                                         |
|     Control, v2.0  | solu |                                         |
|                    | tion |                                         |
| > **HCV (+) C,     | co   |                                         |
| > v2.0**           | ntai |                                         |
|                    | ning |                                         |
|                    | \<0. |                                         |
|                    | 001% |                                         |
|                    | no   |                                         |
|                    | n-in |                                         |
|                    | fect |                                         |
|                    | ious |                                         |
|                    | *in  |                                         |
|                    | vi   |                                         |
|                    | tro* |                                         |
|                    | tra  |                                         |
|                    | nscr |                                         |
|                    | ibed |                                         |
|                    | RNA  |                                         |
|                    | (mi  |                                         |
|                    | crob |                                         |
|                    | ial) |                                         |
|                    | enco |                                         |
|                    | ding |                                         |
|                    | HCV  |                                         |
|                    | se   |                                         |
|                    | quen |                                         |
|                    | ces, |                                         |
|                    | \<0. |                                         |
|                    | 005% |                                         |
|                    | poly |                                         |
|                    | rA   |                                         |
|                    | RNA  |                                         |
|                    | (syn |                                         |
|                    | thet |                                         |
|                    | ic), |                                         |
|                    | EDTA |                                         |
|                    | and  |                                         |
|                    | 0    |                                         |
|                    | .05% |                                         |
|                    | so   |                                         |
|                    | dium |                                         |
|                    | az   |                                         |
|                    | ide, |                                         |
|                    | 8 x  |                                         |
|                    | 0.1  |                                         |
|                    | mL   |                                         |
+--------------------+------+-----------------------------------------+

+-----------------------+----------------------------------------------+
| **AMPLICOR HCV        |                                              |
| Amplification Kit,    |                                              |
| version 2.0**         |                                              |
+-----------------------+----------------------------------------------+
| (7) HCV Master Mix,   | A bicine buffered solution containing 16%    |
|     v2.0              | DMSO, glycerol, \< 0.01% r*Tth* DNA          |
|                       | Polymerase (r*Tth* pol, microbial),          |
| > **HCV MMX, v2.0**   | potassium acetate, \< 0.001% dATP, dCTP,     |
|                       | dGTP, and dUTP, \<0.005% KY78 and KY80       |
|                       | primers (one is biotinylated), \< 0.01%      |
|                       | AmpErase (microbial) and 0.05% sodium       |
|                       | azide, 8 x 0.7 mL                            |
+-----------------------+----------------------------------------------+
| (8) HCV Manganese     | A solution containing \<2% manganese, acetic |
|     Solution, v2.0    | acid, Amaranth dye, 0.05% sodium azide , 8 x |
|                       | 0.1 mL                                       |
| > **HCV Mn^2+^,       |                                              |
| > v2.0**              |                                              |
+-----------------------+----------------------------------------------+
| COBAS AMPLICOR HCV    |                                              |
| Detection Kit,        |                                              |
| version 2.0           |                                              |
+-----------------------+----------------------------------------------+
| (9) HCV Probe         | MES Buffered solution containing \< 0.4%     |
|     Suspension 1,     | Suspension of Dynabeads^®^ (paramagnetic     |
|     v2.0              | particles) coated with HCV-specific          |
|                       | oligonucleotide capture probe (KY150), and   |
| > **CX PS1, v2.0**    | 0.09% sodium azide, 1 x 100 tests            |
+-----------------------+----------------------------------------------+
| (10) HCV Probe        | Sodium phosphate buffered solution           |
|      Suspension 2,    | containing 42.2%% Sodium thiocyanate and     |
|      v2.0             | 0.2% Solubilizer, 1 x 100 tests              |
|                       |                                              |
| > **CX4, v2.0**       |                                              |
+-----------------------+----------------------------------------------+

**Table 2 (continued). Components of the COBAS AMPLICOR** HCV Test,
v2.0

+-----------------------------+----------------------------------------+
| *KIT COMPONENT*             | ### *FORMULATION*                      |
+-----------------------------+----------------------------------------+
| **COBAS AMPLICOR Internal   |                                        |
| Control Detection Kit**     |                                        |
+-----------------------------+----------------------------------------+
| (11) IC Probe Suspension 1  | MES Buffered solution containing \<    |
|                             | 0.35% Suspension of Dynabeads®         |
| > IC PS1                    | (paramagnetic particles) coated with   |
|                             | IC-specific oligonucleotide capture    |
|                             | probe (SK535), 0.09% sodium azide, 1 x |
|                             | 100 tests                              |
+-----------------------------+----------------------------------------+
| (12) IC Probe Suspension 2  | Sodium phosphate buffered solution     |
|                             | containing \<25% Sodium thiocyanate    |
| > **IC4**                   | and 0.2% Solubilizer, 1 x 100 tests    |
+-----------------------------+----------------------------------------+
| **COBAS AMPLICOR Detection  |                                        |
| Reagents Kit**              |                                        |
+-----------------------------+----------------------------------------+
| (13) Denaturation Solution  | A solution containing 1.6% sodium      |
|                             | hydroxide, EDTA and thymol blue , 1 x  |
| > **DN4**                   | 100 tests                              |
+-----------------------------+----------------------------------------+
| (14) Avidin-Horseradish     | A Tris-HCl buffered solution           |
|      Peroxidase Conjugate   | containing \<0.001% avidin-horseradish |
|                             | peroxidase conjugate, bovine serum     |
| > **CN4**                   | albumin (mammalian), Emulsit 25, 0.1%  |
|                             | phenol, 1% ProClin^^ 150,\            |
|                             | 1 x 100 tests                          |
+-----------------------------+----------------------------------------+
| (15) Substrate A            | A citrate solution containing 0.01%    |
|                             | hydrogen peroxide and 0.1% ProClin^^  |
| > **SB3**                   | 150, 5 x 75 tests                      |
+-----------------------------+----------------------------------------+
| (16) Substrate B            | Contains 0.1%                          |
|                             | 3,3′,5,5′-tetramethylbenzidine (TMB)   |
| > **SB**                    | in 40% dimethylformamide (DMF), 5 x    |
|                             | 5mL                                    |
+-----------------------------+----------------------------------------+
| **COBAS AMPLICOR Conjugate  |                                        |
| Detection Reagent**         |                                        |
+-----------------------------+----------------------------------------+
| (17) Avidin-Horseradish     | A Tris-HCl buffered solution           |
|      Peroxidase Conjugate   | containing \<0.001% avidin-horseradish |
|                             | peroxidase conjugate, bovine serum     |
| > **CN4**                   | albumin (mammalian), Emulsit 25, 0.1%  |
|                             | phenol, 1% ProClin^^ 150,\            |
|                             | 2 x 100 tests                          |
+-----------------------------+----------------------------------------+
| **COBAS AMPLICOR Wash       |                                        |
| Buffer**                    |                                        |
+-----------------------------+----------------------------------------+
| (18) 10X-Wash Concentrate   | A solution containing \<2% phosphate   |
|                             | buffer, \<9% sodium chloride, EDTA,    |
| > **WB**                    | \<2% detergent and 0.5% ProClin 300,\  |
|                             | 2 x 250 tests                          |
+-----------------------------+----------------------------------------+

**6. Alternative Practices and Procedures**

6.1. Biochemical Methods

> ***Alanine aminotransferase (ALT)*** is a marker of hepatocellular
> damage and its concentration in serum is often elevated during viral
> hepatitis. Prior to identification of HCV and subsequent development
> of tests for detecting anti-HCV, ALT was used as a surrogate marker
> for "parenterally transmitted non-A, non-B" hepatitis. As such, ALT
> was a non-specific test for HCV infection. More recently, and in the
> absence of a more specific or virologic test, changes in ALT
> concentration were used to assess effectiveness of antiviral therapy
> for chronic hepatitis C. While it is still used in assessing severity
> of chronic hepatitis C without therapy, ALT concentration poorly
> predicts the presence or absence of histopathologic changes in the
> liver.

6.2. Serologic or Virologic Methods

> Currently, there are only a few methods for detecting evidence of HCV
> infection. Such evidence can be indirect (e.g., detection of an
> immunologic response) or direct (e.g., detection of the virus or its
> macromolecules).

6.2.1. Testing for Antibodies to HCV (anti-HCV)

> Anti-HCV assays yield indirect evidence of HCV infection but cannot
> determine if the infection is active (i.e., HCV is actively
> replicating) or inactive. Furthermore, current antibody tests do not
> yield temporal information: results from a single specimen cannot be
> used to determine if an infection is acute or chronic.
>
> ***Enzyme immunoassay (EIA)*** is the most widely used method for
> detecting evidence of HCV infection and is usually a first step in
> laboratory diagnosis of hepatitis C. Current EIAs (FDA-licensed as
> version 2 or 3) detect antibodies to antigens representing core
> (nucleocapsid) and certain non-structural HCV proteins. EIAs are
> readily performed but less than optimally specific. Therefore, package
> inserts for currently licensed EIAs advise supplemental testing to
> ensure that a "repeatedly reactive" result accurately indicates
> detection of antibodies to HCV.
>
> ***Strip immunoassay (SIA)*** is the most widely used method for
> supplemental detection of antibody evidence of HCV infection and,
> currently, version 2 is licensed. CDC and its consultants have also
> recommended using appropriate HCV RNA assays for supplemental evidence
> of infection (see below; Virologic Methods).

6.2.2. Virologic Methods

> ***Virus isolation (propagation)*** cannot currently be used for
> clinical specimens: HCV is not known to replicate in cell culture. It
> is also not practical to inoculate susceptible animals (the only
> well-characterized model of HCV infection uses chimpanzees, *Pan
> troglodytes*).
>
> ***Nucleic-acid detection:*** during recent years, analytically
> sensitive methods for detecting DNA or RNA were developed that have
> enabled practical detection or quantitation of the RNA genome inside
> HCV particles that circulate in the blood. These methods have been
> widely used for research applications, including detection of HCV RNA.
> Many are commercially available, often as generic "kits" that a
> laboratory can adapt for detection of particular analyte like HCV RNA.
>
> All such methods are based on hybridization between reagent nucleic
> acid molecules and DNA or RNA "target" in the sample and subsequent
> production of an identifiable "signal." These methods are frequently
> categorized as "nucleic-acid amplification tests" (NAT), terminology
> that is somewhat misleading because of wide variation in technological
> approaches and in analytical sensitivity. Typically, "target
> amplification" (polymerase-catalyzed production of nucleic acid
> representing the target, followed by generation of
> production-indicating signal) is analytically more sensitive than
> "signal amplification" (enhanced production of signal that indicates
> direct hybridization with target). Examples of target amplification
> methods include PCR, nucleic acid sequence based amplification
> (NASBA), transcription mediated amplification (TMA), self-sustained
> sequence replication (3SR, a non-commercial predecessor to NASBA and
> TMA), strand displacement amplification (SDA), ligase chain reaction
> (LCR), and Qβ replicase (QβR). Examples of target amplification
> methods include branched chain DNA (bDNA) and hybrid capture (HC).
>
> Assays for HCV RNA in the blood are intended to provide information
> about the viremic status of HCV-infected individuals, as demonstrated
> in peer-reviewed scientific literature. Other literature suggests that
> HCV viremia is a reliable marker of active HCV replication in the
> liver. Therefore, HCV RNA assays have been used in research for direct
> evidence of active HCV infection, in diagnostic or monitoring modes.
> The monitoring mode includes assessment of antiviral-therapy effects.
>
> Several HCV RNA assays are commercially available but, other than the
> COBAS AMPLICOR HCV Test, v2.0 and AMPLICOR^®^ HCV Test, v2.0, none
> have been FDA-approved for in vitro diagnostic use. Furthermore, none
> are FDA-approved for monitoring HCV infections (including monitoring
> of response to therapy for hepatitis C) or FDA-licensed for use in
> determining safety of blood, blood products, or tissue. Like the COBAS
> AMPLICOR HCV Test, v2.0 and AMPLICOR^®^ HCV Test, v2.0, other assays
> for HCV RNA often use reagent nucleic acids representing 5\' UTR
> sequences that are highly conserved among HCV genotypes (to optimize
> sensitivity) but distinct from other known nucleotide sequences (in an
> effort to optimize specificity).

**7. Marketing History**

> The COBAS AMPLICOR HCV Test, v2.0 has not been withdrawn from
> marketing in any country for reasons related to safety or
> effectiveness. It is currently available for commercial marketing in
> the following countries:

+---------------------+---------------------+--------------------------+
| -   Argentina       | -   Iceland         | -   Pakistan             |
+---------------------+---------------------+--------------------------+
| -   Austria         | -   India           | -   Paraguay             |
+---------------------+---------------------+--------------------------+
| -   Australia       | -   Indonesia       | -   Philippines          |
+---------------------+---------------------+--------------------------+
| -   Belarus         | -   Ireland         | -   Poland               |
+---------------------+---------------------+--------------------------+
| -   Belgium         | -   Israel          | -   Qatar                |
+---------------------+---------------------+--------------------------+
| -   Chile           | -   Japan           | -   Russia               |
+---------------------+---------------------+--------------------------+
| -   Cyprus          | -   Korea           | -   Saudi Arabia         |
+---------------------+---------------------+--------------------------+
| -   Czech Republic  | -   Kuwait          | -   Slovenia             |
+---------------------+---------------------+--------------------------+
| -   Denmark         | -   Lebanon         | -   South Africa         |
+---------------------+---------------------+--------------------------+
| -   Ecuador         | -   Luxembourg      | -   Spain                |
+---------------------+---------------------+--------------------------+
| -   Egypt           | -   Malaysia        | -   Sweden               |
+---------------------+---------------------+--------------------------+
| -   Finland         | -   Malta           | -   Switzerland          |
+---------------------+---------------------+--------------------------+
| -   France          | -   Mexico          | -   Syria                |
+---------------------+---------------------+--------------------------+
| -   Germany         | -   Netherlands     | -   Turkey               |
+---------------------+---------------------+--------------------------+
| -   Greece          | -   New Zealand     | -   United Arab Emirates |
+---------------------+---------------------+--------------------------+
| -   Hong Kong       | -   Norway          | -   United Kingdom       |
+---------------------+---------------------+--------------------------+
| -   Hungary         | -   Oman            | -   Venezuela            |
+---------------------+---------------------+--------------------------+

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

8.1. Potential Adverse Effects for Patients

> Possibilities for erroneous test results are inherent in all *in
> vitro* diagnostic products. An erroneous result can then lead to an
> adverse effect for the tested patient. Any current test for evidence
> of HCV infection, including those for detecting antibodies to HCV, can
> yield an inaccurate result, or a result that is inaccurately reported
> to the physician or inaccurately interpreted by the physician. The
> possibility of an erroneous COBAS AMPLICOR HCV Test, v2.0 result
> exists due to poor specimen quality (e.g., sample contamination or
> inadequate storage), potential malfunction of a test component, an
> equipment error, or an operator error. Because HCV RNA is the only
> current practical marker of active infection, an inaccurate result or
> interpretation can adversely affect a patient unless other information
> enables the physician to recognize the inaccuracy.
>
> A ***False Positive*** result (i.e., a COBAS AMPLICOR HCV Test, v2.0
> Positive result when HCV RNA was not present in the specimen) could
> lead to a misdiagnosis of active HCV infection. This misdiagnosis
> could lead to unnecessary diagnostic procedures (e.g., liver biopsy,
> which has associated risks and discomfort) or psychological trauma to
> the patient (e.g., concern about a chronic infection that can have
> severe disease consequences). While misdiagnosis could also lead to
> unnecessary treatment (current pharmacologic regimens for chronic
> hepatitis C are less than optimally efficacious and are associated
> with significant side effects), current practice recommendations
> include additional HCV RNA testing (quantitative and genotyping) for
> most patients before therapy begins.
>
> A ***False Negative*** result (i.e., a COBAS AMPLICOR HCV Test, v2.0
> Negative result when HCV RNA was present) could lead to a misdiagnosis
> of cleared, or inactive, HCV infection. This misdiagnosis could lead
> to unnecessary procedures in consideration of other diagnoses; a delay
> in, or failure to perform, appropriate additional diagnostic
> procedures (e.g., to determine the state of active HCV infection or
> severity of HCV-associated liver disease); or a delay in, or failure
> to provide, appropriate treatment.
>
> Data submitted by RMS and in peer-reviewed scientific literature
> provided reasonable assurance of COBAS AMPLICOR HCV Test, v2.0 safety
> and effectiveness. That is, adverse effects could be severe but the
> potential for a false result appeared to be very low (and exceeded by
> the likelihood of benefit from an accurate result).

8.2. Potential Adverse Effects for Laboratory Personnel

> There is also potential for adverse effects, from a COBAS AMPLICOR HCV
> Test, v2.0 component, on the health of laboratory personnel performing
> this assay. One component, Negative Plasma (Human), should be
> considered potentially infectious because it contains human-source
> materials. However, before the component is manufactured, these
> materials must be non-reactive for HBsAg and for antibodies to HCV,
> HIV-1, and HIV-2.
>
> This potential risk occurs with many other *in vitro* diagnostic
> products. Appropriate warnings are included in COBAS AMPLICOR HCV
> Test, v2.0 vial and kit labels and in the Method Manual (Package
> Insert). Most laboratory personnel implement standard good laboratory
> practices and are protected from hepatitis B due to vaccination or
> past infection. Therefore, this risk is well known to laboratory
> personnel and its potential is considered to be minimal.

**9. Summary of Non-Clinical Studies**

> RMS laboratories performed studies to assess analytical sensitivity
> and specificity of the COBAS AMPLICOR HCV Test, v2.0. Those studies
> that contributed to the determination of safety and effectiveness are
> summarized in the Non-Clinical Performance section of the Method
> Manual. Certain findings are also summarized below.

9.1. Limits of Detection (LODs)

> LODs were determined by testing multiple samples containing very low
> concentrations of the WHO International Standard for HCV genotype 1
> RNA\*. This WHO standard was diluted in HCV-negative serum and in
> HCV-negative EDTA plasma to concentrations of 100, 75, 60 and 50
> IU/mL. Each concentration was tested with the COBAS AMPLICOR HCV Test,
> v2.0 in replicates ranging from 80 to 120. LODs were determined as the
> lowest HCV RNA concentration that resulted in ≥ 95% of results
> yielding A~660~ ≥ 1.0, the cutoff for Positive results with the COBAS
> AMPLICOR HCV Test, v2.0. By this criterion, the LOD for HCV RNA in
> serum was 2.0 log~10~ IU/mL, or 100 IU/mL. The LOD for HCV RNA in EDTA
> plasma was 1.8 log~10~ IU/mL, or 60 IU/mL (although 75 IU/mL yielded
> 94% Positive results, \> 95% of all results were Positive for HCV RNA
> ≥ 60 IU/mL).
>
> \*The WHO International Standard for HCV genotype 1 RNA is available,
> as NIBSC code 96/790, from the National Institute for Biological
> Standards and Control, London, UK. IU designates International Units
> of this standard, which contains 10^5^ IU/mL of genotype 1 HCV RNA as
> defined by consensus studies.
>
> Here and in the COBAS AMPLICOR HCV Test, v2.0 Method Manual, *IU*
> designates virion or subgenomic HCV RNA that has been quantified with
> reference to the WHO Standard. *IU*/mL affords a standardized approach
> to indicating HCV RNA concentration but it is not known if *IU*/mL
> accurately reflects the HCV RNA concentration of any particular
> specimen. Available data indicate that 1 IU corresponds to \> 1 HCV
> RNA molecule and that this number of molecules varies according to
> quantifying methods (which are imprecise with a single determination)
> and other variables:

  --------------- ------------ --------------- ------------------ ------------------
  **Source of     **Type of    **How           **Quantifier**     **Conversion
  data**          RNA**        quantified**                       factor**

  Consensus       HCV (virion) End-point       Copies             1 IU ≈ 1.8 copies
                               dilution:                          
                               qualitative                        
                               assays                             

                               Quantitative    Copies or genome   1 IU ≈ 6.6 copies
                               assays          equivalents        

  Roche Molecular Subgenomic   UV spectroscopy A~260~-molecules   1 IU ≈ 2.7
  Systems         transcript                                      A~260~-molecules
  (unpublished)   of cloned                                       (95% CI, 2.6-2.8)
                  HCV cDNA                                        
  --------------- ------------ --------------- ------------------ ------------------

> While RMS has demonstrated similar conversion factors for certain RNAs
> representing HCV genotypes 2-6, it is not known if quantitation with
> reference to the WHO Standard is affected by genotype, strain
> characteristics (including RNA structure and quasispecies), or
> concentration of HCV RNA. For example, IU-to-copy ratios for low
> concentrations of HCV RNA may be different than those for high
> concentrations of HCV RNA.
>
> Samples yielding a result in the Equivocal Zone were not repeat tested
> (per Interpretation of Results section of the Package Insert), so a
> final result was not obtained for these samples. If repeat testing
> were done, it is reasonable to assume that frequencies of Positive
> results would increase, possibly resulting in lower LODs. By excluding
> Equivocal results, for example, the 95% threshold of Positive results
> was reached at 1.8 log~10~ IU/mL (60 IU/mL) for serum and 1.7 log~10~
> IU/mL (50 IU/mL) for EDTA plasma.

9.2. Detection of HCV Genotypes

> Quantified subgenomic RNAs, transcribed from cloned cDNAs, were used
> to approximate analytical sensitivity of the COBAS AMPLICOR HCV Test,
> v2.0 for five HCV genotypes: 1, 2, 3, 4, and 5. (Genotypes of seven
> viruses, including two representatives each of genotypes 1 and 2, were
> determined by using research methods that have not been evaluated or
> approved by FDA; however, genotyping by many such methods is generally
> recognized to be accurate whereas many subtyping techniques vary in
> accuracy.) Each RNA transcript, consisting of the 5\'-untranslated and
> core regions, was quantified by spectrophotometry (A~260~) and then
> diluted to three different concentrations in HCV Specimen Diluent,
> v2.0. Twenty-four replicates of each concentration were tested by the
> COBAS AMPLICOR HCV Test, v2.0. The number of Positive results at each
> concentration was determined. Positive result frequencies were similar
> for these subgenomic RNAs representing genotypes 1--5.
>
> Another study evaluated the sensitivity of the COBAS AMPLICOR HCV
> Test, v2.0 under research laboratory conditions to determine if it
> would yield Positive results with 87 clinical specimens containing HCV
> strains that represented the six recognized genotypes. The majority of
> these specimens were from patients in an antiviral efficacy study and
> were assumed to contain HCV RNA concentrations that were
> representative of those in patients for whom the COBAS AMPLICOR HCV
> Test, v2.0 is indicated. Among the 87 specimens, 86 yielded Positive
> results and one genotype 4 specimen yielded an Equivocal result.

9.3. Cross Contamination

> Two studies were done under controlled laboratory conditions to assess
> the potential cross contamination rate of the COBAS AMPLICOR HCV Test,
> v2.0. In 25 runs of the COBAS AMPLICOR HCV Test, v2.0, multiple
> operators tested replicates of plasma specimens containing
> clinically-pertinent concentrations of HCV RNA (6.5 log~10~ *IU*/mL)
> that alternated with replicates of an HCV-negative specimen. Combined
> results indicated a cross contamination rate of 0.7% (2/273).
>
> The potential for cross contamination was also examined in two
> reproducibility studies performed under clinical laboratory conditions
> with lower concentrations of HCV RNA. These studies demonstrated that
> cross contamination did not occur at \[HCV RNA\] = 2.3 log~10~ *IU*/mL
> with \>530 HCV-negative specimens and at \[HCV RNA\] ≤ 4.7 log~10~
> *IU*/mL with 171 specimens.

9.4. Cross-reactivity with or Interference from Other Microorganisms

> The COBAS AMPLICOR HCV Test, v2.0 was evaluated by testing for
> potential cross-reactivity with, or interference from, certain
> pathogenic microorganisms and normal epidermal microflora that could
> be present in specimens. Twenty-five specimens that contained virus
> (adenoviruses, enteroviruses, herpesviruses, or retroviruses) and four
> that contained bacteria (*Propionibacterium acnes*, *Staphylococcus
> aureus*, *epidermidis*) yielded Negative HCV and Positive HCV IC
> results with the COBAS AMPLICOR HCV Test, v2.0.
>
> The assay was also evaluated for cross-reactivity by testing specimens
> that represented certain manifestations of infection with *Hepatitis A
> virus* (HAV) or *Hepatitis B virus* (HBV). Analytes known to be
> present in these specimens were HAV, IgM antibodies to HAV, HBsAg, or
> HBsAg with HBeAg. All tested serum and plasma specimens yielded
> Negative COBAS AMPLICOR HCV Test, v2.0 results.

9.5. Endogenous Substances

> Serum specimens containing elevated concentrations of albumin,
> bilirubin, hemoglobin, triglycerides, and immunoglobulins were tested
> for interference with the COBAS AMPLICOR HCV Test, v2.0. These
> specimens were tested neat or after spiking with a near-LOD
> concentration of HCV RNA (100 *IU*/mL). Each specimen was tested in
> triplicate.
>
> Specimens not spiked with HCV yielded Negative results, except for 2
> of 11 specimens that contained elevated or nearly elevated levels of
> bilirubin. Negative COBAS AMPLICOR HCV Test, v2.0 results from the
> other 9 elevated-bilirubin specimens indicated lack of interference
> leading to False Positive results.
>
> All three aliquots of an HCV-spiked, high IgG (8610 mg/dL) specimen
> yielded Equivocal results. For one other HCV-spiked specimens with
> elevated concentrations of IgG, one of the three aliquots yielded an
> Equivocal result.

9.6. Therapeutic Drugs

> Drugs for infectious diseases, or for several conditions associated
> with hepatitis C therapy, were evaluated for their potential to
> interfere with the COBAS AMPLICOR HCV Test, v2.0. Among the former
> were drugs for hepatitis C, hepatitis B, HIV, influenza A, or
> cytomegalovirus-associated syndromes. Each drug was spiked to two
> plasma concentrations, peak and 3-times peak (1X and 3X C~max~), into
> plasma that was contained HCV at near-LOD (100 *IU*/ml) or was HCV
> RNA-negative. Specimens were tested in triplicate, with and without
> spiked drugs. Evaluated drugs did not yield False Positive or False
> Negative results. At each drug concentration, all HCV-containing (100
> *IU*/ml) specimens yielded Positive results except for one aliquot
> that contained Crixivan at 1X C~max~, which yielded an Equivocal
> result.

**10. Summary of Clinical Studies**

> These studies are summarized in the Expected Values and Clinical
> Evaluation sections of the COBAS AMPLICOR^TM^ HCV Test, v2.0 Package
> Insert. Certain findings are also summarized below.

10.1. Specimens from Characterized Patients

> A prospective study was conducted at four US hepatology centers to
> evaluate clinical performance of the COBAS AMPLICOR HCV Test, v2.0
> against results of testing for anti-HCV and against a combination of
> anti-HCV results, serum ALT levels, and histologic findings in liver
> tissue. Studied patients were being investigated for evidence of liver
> disease, infection with HCV, or both. Patients were excluded if they
> had undergone liver transplantation or received antiviral therapy for
> Hepatitis C within 6 months of study screening. While HCV RNA was not
> characterized via quantification or genotyping, the patients were
> assumed to represent US populations with active HCV infection and
> chronic liver disease; i.e., range of HCV RNA concentrations (majority
> likely \> 10^4^ *IU*/mL) and HCV genotypes (majority of genotype 1
> viruses, with genotypes 2 and 3 comprising most of the remainder).

10.2. Expected Values

> ***Specimens from patients with antibody evidence of HCV infection,
> not treated for hepatitis C:*** during the clinical study, the vast
> majority of such specimens yielded COBAS AMPLICOR HCV Test, v2.0
> absorbance values (A~660~ ≥ 3.0) much greater than those of the
> Equivocal Zone (0.15 ≤ A~660~ \<1.0): 89% of frozen or refrigerated
> serum and 90% of frozen EDTA plasma. Few specimens required repeat
> testing because of a Potentially Inhibited result (1% of frozen, 0% of
> refrigerated serum and 2% of frozen EDTA plasma) or an Equivocal Zone
> result (0.9% of frozen, 0% of refrigerated serum and 1% of frozen EDTA
> plasma).
>
> ***Run failures***: among 200 runs of the COBAS AMPLICOR HCV Test,
> v2.0 during the clinical study, 10 (5%) failed because Positive
> Controls were out of range (A~660~ \<1.0) and 7 (4%) failed because
> Negative Controls were out of range (A~660~ ≥ 0.1). In addition, there
> were 4 failed runs due to protocol deviations (i.e., failure to follow
> procedures for specimen handling or correct instrument profile setup)
> and 5 system failures (i.e., COBAS Analyzer failures or incomplete
> runs due to improper instrument set-up or mechanical failure).

10.3. Comparison with Results of Anti-HCV Testing

> Studied specimens were from 848 patients, including 154 who had
> received antiviral therapy for chronic hepatitis C that stopped \> 6
> months before collecting specimens. While these previously treated
> patients were included because those with active infections were
> considered to be virologically representative for determining assay
> performance, their data do not imply COBAS AMPLICOR HCV Test, v2.0
> performance for monitoring HCV-associated disease or response to
> treatment. The 694 untreated patients included \>200 who had negative
> anti-HCV EIA results and/or ALT concentration within reference range
> and no histopathologic evidence of hepatitis; they were studied for
> approximating test specificity, but their data do not imply
> performance for testing of individuals without liver disease or
> antibody evidence of HCV infections (see Warnings). In particular,
> Positive COBAS AMPLICOR HCV Test, v2.0 results were obtained for three
> patients who had liver disease other than hepatitis C and for whom
> there was no evidence, or insufficient evidence, to conclude that they
> were actively infected with HCV.
>
> Analysis of data from specimens of serum and EDTA plasma demonstrated
> high concordance with results from anti-HCV testing:

-   For specimens with EIA-repeat reactive (RR) and SIA-positive results
    of anti-HCV testing, the vast majority of COBAS AMPLICOR HCV Test,
    v2.0 results were Positive for both matrices: 94% for serum and 92%
    for EDTA plasma.

-   For EIA-RR / SIA-indeterminate specimens, COBAS AMPLICOR HCV Test,
    v2.0 results were Positive for 66% of serum specimens and 50% of
    EDTA plasma specimens.

-   For EIA-negative samples, the vast majority of COBAS AMPLICOR HCV
    Test, v2.0 results were Negative for both matrices: 97% for serum
    and 96% for plasma.

10.4. Comparison with Anti-HCV, ALT, and Histologic findings

> Test performance was further evaluated in the subset of patients from
> whom liver tissue had been collected in the past. COBAS AMPLICOR HCV
> Test, v2.0 results were compared with the combination of anti-HCV
> results, ALT concentrations, and histologic findings.
>
> ***Patients with elevated ALT levels and histopathologic evidence of
> hepatitis***: COBAS AMPLICOR HCV Test, v2.0 results were Positive for
> 98% of serum and 97% of EDTA plasma specimens that were anti-HCV
> EIA-RR and SIA-positive. COBAS AMPLICOR HCV Test, v2.0 results were
> also Positive for all 8 EIA-RR / SIA-indeterminate specimens. Negative
> results were obtained with 16 out of 17 specimens from EIA-negative
> patients; their histopathologic characteristics were suggestive of
> autoimmune hepatitis, hepatitis B, or other forms of non-C hepatitis.
>
> ***Patients with normal ALT levels and histopathologic evidence of
> hepatitis***: COBAS AMPLICOR HCV Test, v2.0 results were Positive for
> 94% of serum and 87% of EDTA plasma specimens among EIA-RR specimens
> (one of which was SIA-indeterminate; the others were SIA-positive).
> COBAS AMPLICOR HCV Test, v2.0 results were Negative for all 9
> EIA-negative sera from patients with histologic features of hepatitis
> that were suggestive of diseases other than hepatitis C.
>
> ***Patients with elevated ALT levels and no histopathologic evidence
> of hepatitis***: COBAS AMPLICOR HCV Test, v2.0 results were Positive
> for 5 of 7 serum specimens, and both EDTA plasma specimens, with
> EIA-RR / SIA-positive anti-HCV results. COBAS AMPLICOR HCV Test, v2.0
> results were Negative for 97% of EIA-negative serum specimens.
>
> ***Patients with normal ALT levels and no histopathologic evidence of
> hepatitis***: Positive COBAS AMPLICOR HCV Test, v2.0 results were
> obtained from (i) 2 of 4 serum specimen and the single EDTA plasma
> specimen with EIA-RR / SIA-positive results, and (ii) the single
> EIA-RR / SIA-indeterminate sample. COBAS AMPLICOR HCV Test, v2.0
> results were Negative for 97% of serum specimens and for the one EDTA
> plasma specimen from EIA-negative patients. Histopathologic liver
> disease was evident in these cases (but without features of
> hepatitis).

10.5. Reproducibility

> The COBAS AMPLICOR^TM^ HCV Test, v2.0 Method Manual summarizes a
> reproducibility study that was performed in three clinical
> laboratories.

**11. Conclusions Drawn from the Studies**

11.1. Non-Clinical Studies

Analytical Sensitivity

> Regardless of the approach to analyzing submitted data, LODs with the
> WHO International Standard for HCV genotype 1 RNA were ≤2.0 log~10~
> IU/mL, an HCV RNA concentration that is much lower than those expected
> for most patients for whom the COBAS AMPLICOR HCV Test, v2.0 is
> indicated. (Similar data were obtained with other genotype 1 viruses
> in a reproducibility study that was performed in clinical
> laboratories: see Method Manual (Package Insert).
>
> Current technical limitations made it difficult to determine LODs for
> non-1 genotypes: in particular, there are no international reference
> materials for recognized HCV genotypes 2-6 and practical quantifying
> methods have approximately ±0.3 log~10~ imprecision. While these
> limitations prevented LOD determinations from studies with RNAs
> representing non-1 HCV genotypes, the data were not inconsistent with
> those from the WHO Standard (i.e., the data were suggestive of LODs
> within 1 log~10~ of those for genotype 1). These data also
> demonstrated that the COBAS AMPLICOR HCV Test, v2.0 was likely to
> detect HCV genotypes 2-6 at much lower RNA concentrations that those
> expected for most patients for whom the assay is indicated.
>
> These findings are evidence of the safety and effectiveness of the
> COBAS AMPLICOR HCV Test, v2.0 for its indicated use. In addition, data
> from an internationally recognized reference material, the WHO
> International Standard for HCV genotype 1 RNA, is appropriate and
> essential for enabling comparison among HCV RNA assays.
>
> **Analytical Specificity**
>
> **Potential Cross-reactivity or Interference**
>
> Submitted data yielded no evidence of COBAS AMPLICOR HCV Test, v2.0
> cross-reactivity with, or interference from, most of the analytes that
> were tested, including certain microorganisms, serum proteins, and
> pertinent therapeutic drugs.
>
> The tested endogenous substances were shown not to interfere with the
> COBAS AMPLICOR HCV Test, v2.0, with the exception of IgG (4 of 12
> tested aliquots) or triglycerides (3 of 39 aliquots, representing 3
> specimens). Conclusions about these results were tentative because few
> data were submitted for these and other such specimens and because
> Equivocal results are expected for several percent of aliquots when
> the concentration of HCV RNA is near-LOD.These concerns are noted in
> the procedural limitations section.
>
> Certain specimens containing bilirubin might have yielded False
> Positive results. Among 11 specimens tested for analytical specificity
> in the presence of elevated concentrations of bilirubin and without
> spiked HCV, 2 did not yield Negative COBAS AMPLICOR HCV Test, v2.0
> results. It could not be determined if the Positive and Equivocal
> results from these 2 specimens represented accurate detection of HCV
> RNA, because anti-HCV results were not available. These concerns are
> noted in the procedural limitations section. Non-clinical studies of
> the COBAS AMPLICOR HCV Test, v2.0 did not include evaluation of
> potential interference with detection of low HCV RNA concentrations by
> other analytes that often circulate at elevated concentrations during
> viral hepatitis. While there was no apparent effect of ALT on
> frequencies of Positive results during the clinical study, it was
> assumed that most studied patients had \>10^4^ *IU*/mL of HCV RNA;
> furthermore, most specimens were frozen (which reduces concentrations
> of these analytes) before COBAS AMPLICOR HCV Test, v2.0 testing. These
> concerns are noted in the procedural limitations section. While tested
> therapeutic drugs did not appear to interfere with the ability to
> detect HCV RNA, specimens were spiked with each drug and so the study
> did not provide data about drug metabolites that might affect assay
> performance. In addition, non-clinical studies did not evaluate
> potential effects of anti-bacterial or anti-fungal drugs that are
> frequently administered to patients. FDA did not recommend any
> pertinent post-approval studies.
>
> **Cross Contamination**
>
> Cross contamination is a recognized problem for all laboratory assays,
> and particularly those based on target amplification occurred.
> Submitted data indicated infrequent (≤0.7%) cross- contamination rates
> during studies of the COBAS AMPLICOR HCV Test, v2.0.

11.2. Clinical Studies

> **Study of patients with liver disease**
>
> The study of patients with liver disease demonstrated high concordance
> between COBAS AMPLICOR HCV Test, v2.0 results and those from anti-HCV
> testing. There was also high concordance between COBAS AMPLICOR HCV
> Test, v2.0 results and the combination of serologic, biochemical and
> histologic findings. Such concordance was plausible because study data
> indicated that most of the studied patients were likely to have had
> chronic liver disease. Peer-reviewed scientific literature had
> previously demonstrated that (i) most HCV infections persist with
> detectable HCV viremia, particularly in individuals with elevated
> serum concentrations of ALT; and (ii) EIA-negative HCV infections with
> detectable RNA are very unusual, especially in a chronically infected
> population.
>
> Limits of current technology and design of the study made it difficult
> to determine the true frequencies of false COBAS AMPLICOR HCV Test,
> v2.0 results. That is, while there may have been unrecognized False
> Positive and False Negative COBAS AMPLICOR HCV Test, v2.0 results, FDA
> decided that the study data were appropriate for contributing to the
> determination of safety and effectiveness. Most importantly, there is
> no reference assay with known accuracy for detecting HCV RNA.
>
> Study design did not include determination of HCV genotypes or HCV RNA
> concentrations. While it could not be determined if these variables
> affected COBAS AMPLICOR HCV Test, v2.0 performance, FDA recognized the
> absence of reference assays for genotyping or quantifying HCV RNA and
> accepted the assumption that these variables were adequately
> represented among enrolled patients at the four US study sites.
> Furthermore, non-clinical studies demonstrated that any effects of
> genotype were unlikely at the high concentrations of HCV RNA (\>10^4^
> *IU*/mL) that most patients in this study were assumed to have had.
>
> The data from this clinical study demonstrated that COBAS AMPLICOR HCV
> Test, v2.0 yielded Positive results for a very high proportion of
> patients with liver disease and antibody evidence of HCV infection.
> The assay also yielded Negative results for a very high proportion of
> patients who had liver disease without anti-HCV (these data do not
> imply performance for testing of individuals without liver disease or
> antibody evidence of HCV infections). Although the exact proportions
> of false results could not be determined, FDA decided that the data
> provided reasonable assurance of COBAS AMPLICOR HCV Test, v2.0 safety
> and effectiveness for diagnosis of active HCV infection among patients
> with evidence of liver disease and antibody evidence of HCV infection.
> Effectiveness of the COBAS AMPLICOR HCV Test, v2.0 was also supported
> by the low frequencies, during the clinical study, of failed runs and
> of Potentially Inhibited and Equivocal results that require repeat
> testing.
>
> **Reproducibility**
>
> Performance of the COBAS AMPLICOR HCV Test, v2.0 assay was
> reproducible among studied variables that included reagent lots, study
> sites, and days, and sample matrices (See package insert).

11.3. Risk / Benefit Analysis

> The presence of HCV viremia, as determined by detecting HCV RNA, is
> direct evidence of replication of the virus; i.e., active HCV
> infection. Thus, the most significant benefit is that COBAS AMPLICOR
> HCV Test, v2.0 results can provide evidence of active HCV infection
> for patients who have evidence of liver disease and antibody evidence
> of HCV infection. (Antibody evidence of HCV infection is defined here
> as repeatedly reactive results from an anti-HCV EIA alone, or in
> combination with positive or indeterminate results from an anti-HCV
> SIA.)
>
> Available data indicate that the likelihood of benefit, to patients
> tested by the COBAS AMPLICOR HCV Test, v2.0, exceeds any known or
> potential adverse event or risk to patients or laboratory personnel.
>
> The clinical and non-clinical laboratory studies described above
> provided reasonable assurance that the COBAS AMPLICOR HCV Test, v2.0
> can be safely and effectively used for qualitative detection of HCV
> RNA in human serum or EDTA plasma. These studies also provided
> scientific evidence of diagnostic benefits to be gained from FDA
> approval for marketing of the COBAS AMPLICOR HCV Test, v2.0.

11.4. Safety

> As a diagnostic test, the COBAS AMPLICOR^TM^ HCV Test, v2.0 assay
> involves removal of blood from an individual for testing purposes. The
> test, therefore, presents no more safety hazards to and individual
> being tested than other tests where blood is removed.

**12. Panel Recommendations**

> The Microbiology Devices Advisory Panel (MDAP) met on July 28, 2000 to
> consider safety and effectiveness of the COBAS AMPLICOR HCV Test,
> v2.0. MDAP recommended approval subject to the following conditions:

1)  RMS should submit, and FDA should satisfactorily analyze,
    appropriate data for determining the assay's reproducibility.

2)  The Package Insert should include revisions to the intended use,
    warnings, precautions and performance sections.

**13. CDRH Decision**

> CDRH concurred with MDAP recommendations. RMS provided additional
> data, analyses, and peer-reviewed scientific literature to address
> assay reproducibility and other issues. Certain issues were rectified
> by appropriate data that RMS submitted in the Amendments, by FDA
> analyses of RMS data and peer-reviewed scientific literature, or by
> warnings and limitations in the Package Insert (Labeling
> restrictions). Other issues were rectified by deciding that the
> probability of unrecognized inaccurate performance or adverse effects
> was exceeded by public health importance of the first approval for US
> marketing of an HCV RNA assay. The Package Insert was revised by using
> the MDAP recommendations.
>
> On October 6, 2000, the applicant's two manufacturing facilities were
> found to be in compliance with the Quality Systems Regulation (21 CFR
> 820).
>
> Having determined that there was reasonable assurance of safety and
> effectiveness for the COBAS AMPLICOR HCV Test, v2.0, CDRH issued an
> approval order on July 3, 2001.

**14. Approval Specifications**

#### Directions for Use: see Method Manual (Package Insert) and other Labeling

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

Postapproval Requirements and Precautions: See Approval Order.

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