雅培RealTime HCV Genotype⒒

Abbott RealTime

HCV Genotype II

08L21-9051-608500/R1Rx Only

Key to symbols used

Global Trade Item Number

Reference Number

Consult instructions for use In Vitro Diagnostic Medical Device Internal Control Amplification Reagent Pack A Amplification Reagent Pack B Amplification Reagent Pack C Positive Control Negative Control Temperature Limitation

Warning

See REAGENTS section for a full explanation of symbols used in reagent component naming. Customer Service: 1-800-553-7042

Customer Service International: Call your Abbott Representative

This package insert must be read carefully prior to use. Package insert instructions must be followed accordingly. Reliability of assay results cannot be guaranteed if there are any deviations from the instructions in this package insert.

NAME

Abbott RealTime HCV Genotype II

INTENDED USE

The Abbott RealTime HCV Genotype II is an in vitro reverse transcription-polymerase chain reaction

(RT-PCR) assay for use with the Abbott mSample Preparation System reagents and with the Abbott m2000sp and m2000rt instruments for the qualitative identification of hepatitis C virus (HCV) genotypes 1, 1a, 1b, and 2 - 5 in plasma or serum from individuals chronically infected with HCV.

The Abbott RealTime HCV Genotype II is intended for use as an aid in the management of HCV-infected individuals and in guiding the selection of therapeutic treatment indicated for the above listed genotypes. The assay is intended for use on patients who are chronically infected with HCV, are being considered for antiviral treatment, and are positive for HCV RNA.

The Abbott RealTime HCV Genotype II assay is not for screening blood, plasma, serum or tissue donors for HCV.

SUMMARY AND EXPLANATION OF THE TEST

The Hepatitis C virus (HCV), a significant cause of blood-borne hepatitis, is an enveloped virus containing a single-stranded positive sense RNA genome of approximately 9,500 nucleotides.1 It has been identified as the major etiological agent for post-transfusion non-A and non-B hepatitis worldwide. Based on genetic similarity, HCV has been classified into six major genotypes (1 – 6) and numerous subtypes (1a, 1b, etc.).2 HCV genotype impacts the response of HCV-infected patients to peg-interferon/ribavirin combination therapy.3 Before starting combination therapy, it is recommended that the genotype of the infecting HCV isolate be determined so that the patient can receive the most appropriate therapy regimen.4

HCV genotype 1 is the predominant HCV genotype in the United States (73.32%) followed by HCV genotype 2 (13.10%) and HCV genotype 3 (12.13%) and HCV genotype 4 (1.32%). HCV genotypes 5 and 6 each represent less than or equal to 1% of HCV genotype determinations in the US.5

The Abbott RealTime HCV Genotype II Positive Control is standardized against the Second WHO International Standard for Hepatitis C Virus RNA (NIBSC Code 96/798).6

BIOLOGICAL PRINCIPLES OF THE PROCEDURE

The Abbott RealTime HCV Genotype II assay consists of two reagent kits:

Abbott RealTime HCV Genotype II Amplification Reagent Kit

Abbott RealTime HCV Genotype II Control Kit

The Abbott RealTime HCV Genotype II is an in vitro reverse transcription-polymerase chain reaction

(RT-PCR) assay for the identification of the genotype(s) of hepatitis C virus (HCV) in plasma or serum from individuals chronically infected with HCV. The Abbott RealTime HCV Genotype II assay detects genotypes 1, 1a, 1b and 2 – 5 through the use of genotype-specific fluorescent-labeled oligonucleotide probes.

Sample Preparation

The Abbott m2000sp provides automated sample preparation using a magnetic microparticle-based protocol (Abbott mSample Preparation System) to process 0.5 mL samples (ACD-A, CPD, potassium EDTA, or sodium EDTA plasma or serum). During the sample preparation protocol, HCV virions are disrupted by guanidine isothiocyanate, RNA is captured on the magnetic microparticles, inhibitors are removed by washing steps, and RNA is eluted off the microparticles. The bound nucleic acids are eluted and transferred to a 96 deep-well plate. The nucleic acids are then ready for amplification. The Internal Control (IC) is introduced into each specimen at

the beginning of the sample preparation process to demonstrate that the process was completed correctly for each specimen and control.

Amplification Master Mix

The Abbott m2000sp instrument automates the assembly of three amplification master mixes (A, B, and C) by combining the respective Abbott RealTime HCV Genotype II Oligonucleotide Reagent (A, B, or C) with thermostable rTth DNA polymerase enzyme and Activation Reagent. The Abbott m2000sp dispenses the resulting master mixes into the Abbott 96-Well Optical Reaction Plate along with aliquots of the nucleic acid samples prepared by the Abbott m2000sp. Each processed sample is added to one well containing Master Mix A, one well containing Master Mix B, and one well containing Master Mix C.

Amplification

The Abbott RealTime HCV Genotype II assay uses four sets of PCR primers. One set of primers targets a

sequence within the 5′ untranslated region (UTR) of the HCV genome. This primer set is designed to amplify all HCV isolates. The second primer set is designed to amplify the non structural 5b (NS5b) region of genotype 1a. The third HCV primer set is designed to amplify the NS5b region of genotype 1b. By contrast, the IC primer set is designed to amplify a portion of the hydroxypyruvate reductase gene of the pumpkin plant, Cucurbita pepo and is delivered in an Armored RNA® particle that has been diluted in negative human plasma.

During the amplification reaction, the target RNA is converted to complementary DNA (cDNA) by the reverse transcriptase activity of the thermostable rTth DNA polymerase.7 The HCV and IC reverse primers anneal to their respective targets and are extended during a prolonged incubation period. After a denaturation step, in which the temperature of the reaction is raised above the melting point of the double-stranded cDNA:RNA product, a second primer anneals to the cDNA strand and is extended by the DNA polymerase activity of the rTth enzyme to create a double-stranded DNA product.

During each round of thermal cycling, amplification products dissociate to single strands at high temperature allowing primer annealing and extension as the temperature is lowered. Exponential amplification of the

product is achieved through repeated cycling between high and low temperatures, resulting in a billion-fold or greater amplification of target sequences.

Detection

The assay requires three separate reactions to detect genotypes 1, 1a, 1b and 2 – 5:

Reaction A is designed to detect all HCV isolates, type 3 isolates, and subtype 1a isolates.

Reaction B is designed to detect type 1 isolates, type 2 isolates, and subtype 1b isolates.

Reaction C is designed to detect type 4 isolates and type 5 isolates. Reaction Probe A All HCV Isolates FAM

Genotype 3 Subtype 1a B Genotype 1 Genotype 2 Subtype 1b C Genotype Genotype 5

A - C

aInternal Control (IC) Quasar 670a Reference Dye ROXb

b Quasar 670 dye contains the same spectral properties as Cy 5. ROX is a Passive Reference Dye in Oligonucleotide Reagents A, B, and C.

Amplification of both HCV and IC targets take place simultaneously in the same reaction. The HCV genotype-specific probes and IC-specific probe within each reaction are all labeled with different fluorophores, thus allowing for simultaneous detection of HCV genotype-specific and IC-specific amplified products.

During the annealing portion of each amplification cycle, the probes hybridize to their respective amplification target, if present. The 5′ end of each HCV-specific probe is labeled with a fluorescent moiety while the 3′

terminus is labeled with a quenching moiety and a Minor Groove Binder (MGB ) group. In the absence of the HCV target sequences, the probe fluorescence is quenched. In the presence of the HCV target sequences, the probe hybridizes to its complementary sequence. During PCR extension, the 5′ to 3′ exonuclease (or Taqman) activity of the rTth polymerase degrades the hybridized probe into constituent nucleotides thus separating the quencher and the fluorophore allowing fluorescent emission and detection.8

The Abbott m2000rt instrument detects the resultant fluorescence of the different fluorophores in each reaction well after each cycle.

PREVENTION OF NUCLEIC ACID CONTAMINATION

The possibility of nucleic acid contamination is minimized because:

Reverse transcription, PCR amplification, and oligonucleotide hybridization occur in a sealed 96-Well Optical Reaction Plate.

Detection is carried out automatically without the need to open the 96-Well Optical Reaction Plate. Aerosol barrier pipette tips are used for all pipetting. The pipette tips are discarded after use.

Separate dedicated areas are used to perform the Abbott RealTime HCV Genotype II assay. Refer to the

SPECIAL PRECAUTIONS section of this package insert.

REAGENTS

Abbott RealTime HCV Genotype II Amplification Reagent Kit (List No. 08L21-90)

1. Abbott RealTime HCV Genotype II Internal Control (List No. 8L21Y) (2 vials, 1.2 mL per vial)

Less than 0.01% noninfectious Armored RNA with internal control sequences in negative human

plasma. Negative human plasma tested and found to be nonreactive by FDA licensed tests for antibody to HCV, antibody to HIV-1, antibody to HIV-2, and HBsAg. The material is also tested and found to be negative by FDA licensed PCR methods for HIV-1 RNA and HCV RNA. Preservatives: 0.1% ProClin® 300 and 0.15% ProClin 950.

2. Abbott RealTime HCV Genotype II Amplification Reagent Packs (ListNo.08L21)

(Reagent Packs A, B, C / 24 Tests)

(1) Reagent Pack A.

1 bottle (0.9 mL) HCV Genotype II Oligonucleotide Reagent A. Less than 0.1% synthetic

oligonucleotides (6 primers, 5 probes), less than 0.1% dNTPs, and 10.4% dimethylsulfoxide in a

buffered solution with a reference dye. Preservatives: 0.1% ProClin 300 and 0.15% ProClin 950.

1 bottle (0.141 mL) Thermostable rTth Polymerase Enzyme (2.9 to 3.5 Units/µL) in buffered solution. 1 bottle (0.400 mL) Activation Reagent, 30 mM manganese chloride solution.

Preservatives: 0.1% ProClin 300 and 0.15% ProClin 950.

(2) Reagent Pack B.

1 bottle (0.9 mL) HCV Genotype II Oligonucleotide Reagent B. Less than 0.1% synthetic

oligonucleotides (6 primers, 5 probes), less than 0.1% dNTPs, and 10.4% dimethylsulfoxide in a

buffered solution with a reference dye. Preservatives: 0.1% ProClin 300 and 0.15% ProClin 950.

1 bottle (0.141 mL) Thermostable rTth Polymerase Enzyme (2.9 to 3.5 Units/µL) in buffered solution. 1 bottle (0.400 mL) Activation Reagent, 30 mM manganese chloride solution.

Preservatives: 0.1% ProClin 300 and 0.15% ProClin 950. (3) Reagent Pack C.

1 bottle (0.9 mL) HCV Genotype II Oligonucleotide Reagent C. Less than 0.1% synthetic

oligonucleotides (4 primers, 4 probes), less than 0.1% dNTPs, and 10.4% dimethylsulfoxide in a

buffered solution with a reference dye. Preservatives: 0.1% ProClin 300 and 0.15% ProClin 950.

1 bottle (0.141 mL) Thermostable rTth Polymerase Enzyme (2.9 to 3.5 Units/µL) in buffered solution. 1 bottle (0.400 mL) Activation Reagent, 30 mM manganese chloride solution.

Preservatives: 0.1% ProClin 300 and 0.15% ProClin 950.

NOTE: Amplification reagents are intended for single-use only. Residual reagent will remain in the reagent pack bottles after use. Unused reagents should be discarded.

Abbott RealTime HCV Genotype II Control Kit (List No. 08L21-80)

1.

Abbott RealTime HCV Genotype II Negative Control (List No. 8L21Z)

(4 vials, 1.3 mL per vial)

Negative human plasma tested and found to be nonreactive by FDA licensed tests for antibody to HCV, antibody to HIV-1, antibody to HIV-2, and HBsAg. The material is also tested and found to be negative by FDA licensed PCR methods for HIV-1 RNA and HCV RNA. Preservatives: 0.1% ProClin 300 and 0.15% ProClin 950.

Abbott RealTime HCV Genotype II Positive Control (List No. 8L21W)

(4 vials, 1.3 mL per vial)

Noninfectious Armored RNA representing the 5′ untranslated region (5′ UTR) sequences of HCV

genotype 1a and HCV genotype 4 in negative human plasma. Negative human plasma tested and found to be nonreactive by FDA licensed tests for antibody to HCV, antibody to HIV-1, antibody to HIV-2, and HBsAg. The material is also tested and found to be negative by FDA licensed PCR methods for

HIV-1 RNA and HCV RNA. Preservatives: 0.1% ProClin 300 and 0.15% ProClin 950. 2.

NOTE: Control lots can be used interchangeably with amplification reagent kit lots.

WARNINGS AND PRECAUTIONS

In Vitro Diagnostic Medical Device

FOR IN VITRO DIAGNOSTIC USE

The Abbott RealTime HCV Genotype II assay is indicated only for individuals who have been determined to be chronically infected with HCV.

The Abbott RealTime HCV Genotype II assay is not for screening blood, plasma, serum or tissue donors for HCV, or to be used as a diagnostic test to confirm the presence of HCV infection in donated blood, plasma, serum, or tissue.

The Abbott RealTime HCV Genotype II reagents are intended to be used only on the Abbott m2000

System consisting of the Abbott m2000sp for sample processing and the Abbott m2000rt for amplification and detection.

Only use Uracil-N-Glycosylase (UNG) List No. 08L21-66 when performing the Uracil-N-Glycosylase protocol. Do not use kits or reagents beyond expiration date.

If the Abbott m2000sp master mix addition protocol is aborted, seal the Abbott 96-Well Optical Reaction Plate in a sealable plastic bag and dispose according to the Abbott m2000sp Operations Manual, Hazards section, along with the gloves used to handle the plate. Do not import the test order onto the Abbott m2000rt.

The appropriate PCR plate must be selected when samples are loaded into the Abbott m2000rt instrument. NOTE: The Abbott m2000rt protocol must be started within 90 minutes of the initiation of the Master Mix Addition protocol (Assay Protocol Step 12).

If the Abbott m2000rt instrument run is not initiated within 90 minutes, or is interrupted or aborted, seal the Abbott 96-Well Optical Reaction Plate in a sealable plastic bag and dispose according to the Abbott m2000rt Operations Manual along with the gloves used to handle the plate.

Safety Precautions

Refer to the Abbott m2000sp Operations Manual, Hazards Section and the Abbott m2000rt Operations Manual, Hazards Section for instructions on safety precautions.

CAUTION: The Control Kit and Internal Control contain human sourced and/or potentially infectious components. Components sourced from human blood have been tested and found to be nonreactive by FDA licensed tests for antibody to HCV, antibody to HIV-1, antibody to HIV-2, and HBsAg. The

material is also tested and found to be negative by FDA licensed PCR methods for HIV-1 RNA and

HCV RNA. No known test method can offer complete assurance that products derived from human

sources will not transmit infection. Therefore, all human sourced materials should be considered

potentially infectious. It is recommended that these reagents and human specimens be handled as if

infectious, using safe laboratory procedures, such as those outlined in Biosafety in Microbiological and Biomedical Laboratories,9 OSHA Standard on Bloodborne Pathogens,10 CLSI Document M29-A3,11

and other appropriate biosafety practices.11,12 Therefore, all human sourced materials should be

considered potentially infectious.

The Abbott RealTime HCV Genotype II Control Kit, Internal Control, HCV Oligonucleotide Reagents A, B, and C, and Activation Reagent contain a 3:1mixture of 5-chloro-2- methyl-4-isothiazolin-3-one and 2-methyl-4-isothiazolin-3-one which are components of ProClin.

H319

H317

P261

P264

P280

P272

P305+P351+P338

P302+P352

P333+P313

P337+P313

P363

P501 Causes serious eye irritation. May cause an allergic skin reaction. Avoid breathing mist / vapors / spray. Wash hands thoroughly after handling. Wear protective gloves / protective clothing / eye protection. Contaminated work clothing should not be allowed out of the workplace. IF IN EYES: Rinse cautiously with water for several minutes. Remove contact lenses, if present and easy to do. Continue rinsing. IF ON SKIN: Wash with plenty of water. If skin irritation or rash occurs: Get medical advice / attention. If eye irritation persists: get medical advice / attention. Wash contaminated clothing before reuse. This material and its container must be disposed of in a safe way.

These precautions include, but are not limited to, the following:

Wear gloves when handling specimens or reagents.

Do not pipette by mouth.

Do not eat, drink, smoke, apply cosmetics, or handle contact lenses in areas where these materials are handled.

Clean and disinfect spills of specimens by using a tuberculocidal disinfectant such as 1.0% sodium hypochlorite or other suitable disinfectant.13,14

Decontaminate and dispose of all specimens, reagents, and other potentially contaminated materials in accordance with local, state, and federal regulations.15,16

Special Precautions

Handling Precautions

The Abbott RealTime HCV Genotype II assay is only for use with human serum or plasma (ACD-A, CPD, potassium EDTA, or sodium EDTA) specimens that have been handled and stored in capped tubes as described in the SPECIMEN COLLECTION, STORAGE, AND TRANSPORT TO THE TEST SITE section. During preparation of samples, compliance with good laboratory practices is essential to minimize the risk of cross-contamination between samples, and the inadvertent introduction of ribonucleases (RNases) into samples during and after the extraction procedure. Proper aseptic technique should always be used when working with RNA.

Amplification reactions such as PCR are sensitive to accidental introduction of product from previous

amplification reactions. Incorrect results could occur if either the clinical specimen or the RealTime reagents used in the amplification step become contaminated by accidental introduction of even a few molecules of amplification product. Measures to reduce the risk of contamination in the laboratory include physically separating the activities involved in performing PCR and complying with good laboratory practices.

Work Areas

The m2000sp and the m2000rt instruments may be operated in the same location. Use two dedicated areas within the laboratory for performing the Abbott RealTime HCV Genotype II assay.

The Sample Preparation Area is dedicated to processing samples (specimens and Abbott RealTime HCV Genotype II Controls), and to adding processed samples and controls to the Abbott 96-Well

Optical Reaction Plate. All reagents used in the Sample Preparation Area should remain in this

dedicated area at all times. Laboratory coats, pipettes, pipette tips, and vortexers used in the

Sample Preparation Area must remain in this area and not be moved to the Amplification Area. Do not bring amplification product into the Sample Preparation Area.

The Amplification Area is dedicated to the amplification and detection of amplified product.

Laboratory coats and equipment used in the Amplification Area must remain in this area and not be

moved to the Sample Preparation Area.

Components contained within a kit are intended to be used together. Do not mix components from different kit lots. For example, do not use the negative control from control kit lot X with the positive controls from control kit lot Y. Additionally, do not mix and match Amplification Reagent Packs A, B, and C from different amplification kit lots.

Do not use kits or reagents beyond expiration date.

Work areas and instrument platforms must be considered potential sources of contamination. Change gloves after contact with potential contaminants (such as specimens, eluates, and/or amplified product) before handling unopened reagents, negative control, positive controls, or specimens. Refer to the Abbott m2000sp and m2000rt Operations Manuals for instructions on instrument cleaning procedures.

If the Abbott m2000sp instrument run is aborted, dispose of all commodities and reagents according to the Abbott m2000sp Operations Manual. If the Abbott m2000sp master mix addition protocol is aborted, seal the Abbott 96-Well Optical Reaction Plate in a sealable plastic bag and dispose according to the Abbott m2000sp Operations Manual, Hazards Section, along with the gloves used to handle the plate.

If the Abbott m2000rt instrument run is interrupted or aborted, seal the Abbott 96-Well Optical Reaction Plate in a sealable plastic bag and dispose according to the Abbott m2000rt Operations Manual along with the gloves used to handle the plate.

Decontaminate and dispose of all specimens, reagents, and other potentially biohazardous materials in accordance with local, state, and federal regulations.15,16 All materials should be handled in a manner that minimizes the chance of potential contamination of the work area. NOTE: Autoclaving the sealed Abbott 96-Well Optical Reaction Plate will not degrade the amplified product and may contribute to the release of the amplified product by opening the sealed plate. The laboratory area can become contaminated with amplified product if the waste materials are not carefully handled and contained before and after processing.

Aerosol Containment

To reduce the risk of nucleic acid contamination due to aerosols formed during manual pipetting, aerosol barrier pipette tips must be used for all manual pipetting. The pipette tips must be used only one time. Clean and disinfect spills of specimens and reagents as stated in the Abbott m2000sp and Abbott m2000rt Operations Manuals.

Contamination and Inhibition

The following precautions should be observed to minimize the risks of RNase contamination, cross-

contamination between samples, and inhibition:

Wear appropriate personal protective equipment at all times.

Use powder-free gloves.

Change gloves after having contact with potential contaminants (such as specimens, eluates, and/or amplified product).

To reduce the risk of nucleic acid contamination due to aerosols formed during pipetting, pipettes with aerosol barrier tips must be used for all sample and IC reagent pipetting. The length of the tip should be sufficient to prevent contamination of the pipette barrel. While pipetting, care should be taken to avoid touching the pipette barrel to the inside surface of the sample tube or container. The use of extended

aerosol barrier pipette tips is recommended.

Change aerosol barrier pipette tips between ALL manual liquid transfers.

Clean and disinfect spills of specimens and reagents as stated in the Abbott m2000sp and the Abbott m2000rt Operations Manuals, Hazards section.

Replace any empty or partially used 200 μL and 1000 μL disposable tip trays with full trays before every run.

The Abbott mSample Preparation System (4 x 24 Preps) reagents are single use only. Use new reagent vessels, reaction vessels, and newly opened reagents for every new Abbott RealTime HCV Genotype II assay run. At the end of each run, discard all remaining reagents from the Abbott m2000sp worktable as stated in the Abbott m2000sp Operations Manual and the Abbott mSample Preparation System

(4 x 24 Preps) product information sheet.

Contamination From External dU-Containing Amplified Product

Laboratories that use or have used HCV amplification assays that include post-PCR processing of the amplified product may be contaminated by dU-containing amplified product. Such contamination may cause inaccurate results in the Abbott RealTime HCV Genotype II assay. Refer to the Monitoring the Laboratory for the Presence of Amplification Product section of this package insert.

When negative controls persistently generate a HCV genotype result, are invalid or where contamination with dU containing HCV amplified product is likely to have occurred, it is recommended that the laboratory use the Uracil-N-Glycosylase (UNG) (List No. 08L21-66) contamination control procedure if decontamination of the laboratory is unsuccessful.

STORAGE INSTRUCTIONS

Abbott RealTim

e HCV Genotype II Amplification Reagent Kit (List No. 08L21-90)

The Abbott RealTime HCV Genotype II Amplification Reagent Packs and Internal

Control vials must be stored at –25 to –15°C when not in use. Care must be taken to separate the Abbott RealTime HCV Genotype II Amplification Reagent Packs that are in

use from direct contact with samples and controls.

Abbott RealTim

e HCV Genotype II Control Kit (List No. 08L21-80)

The Abbott RealTime HCV Genotype II Negative and Positive Controls must be stored

at –25 to –15°C.

INDICATION OF INSTABILITY OR DETERIORATION OF REAGENTS

When a positive or negative control value is out of the expected range, it may indicate deterioration of the reagents. Associated test results are invalid and samples must be retested.

SPECIMEN COLLECTION, STORAGE, AND TRANSPORT TO THE TEST SITE Specimen Collection and Storage

Human serum or plasma (ACD-A, CPD, potassium EDTA, or sodium EDTA) specimens may be used with the Abbott RealTime HCV Genotype II assay. Follow the manufacturer’s instructions for processing collection tubes.

Freshly drawn specimens (whole blood) may be held at 2 to 30°C for up to 6 hours

At 15 to 30°C for up to 24 hours At 2 to 8°C for up to 3 days At –25 to –15°C for up to 60 days At –70°C for up to 60 days priorto centrifugation. After centrifugation, remove serum or plasma from cells. Serum or plasma specimens may be stored:

Multiple freeze/thaw cycles should be avoided. If frozen, thaw specimens at 15 to 30°C or at 2 to 8°C. Once thawed, if specimens are not being processed immediately, they can be stored at 2 to 8°C for up to 6 hours. Specimens showing particulate matter or turbidity should be clarified by centrifugation at 2000 g for 5 minutes prior to testing.

Specimen Transport

Ship specimens frozen on dry ice. Specimens should be packaged and labeled in compliance with applicable state and federal regulations covering the transport of clinical specimens and etiologic agents/infectious substances.

INSTRUMENT PROCEDURE

The Abbott RealTime HCV Genotype II application file must be installed on the Abbott m2000sp and Abbott m2000rt instruments from the Abbott RealTime HCV Genotype II m2000 System Combined Application CD-ROM prior to performing the assay. For detailed information on application file installation, refer to the Abbott m2000sp and m2000rt Operations Manuals, Operating Instructions section.

ABBOTT REALTIME HCV GENOTYPE II ASSAY PROCEDURE

Materials Provided

Abbott RealTime HCV Genotype II Amplification Reagent Kit (List No. 08L21-90)

Materials Required But Not Provided

Abbott RealTime HCV Genotype II Control Kit (List No. 08L21-80) Abbott m2000sp Instrument

Abbott mSample Preparation System (4 x 24 Preps) (List No. 04J70-24)

Abbott RealTime HCV Genotype II m2000 System Combined Application CD-ROM

Uracil-N-Glycosylase (UNG) (List No. 08L21-66) protocol

5 mL Reaction Vessels

200 mL Reagent Vessels

Transport Tubes (Master Mix Vials)

Abbott 96-Well Optical Reaction Plate

Abbott 96-Deep-Well Plate

Abbott Splash-Free Support Base

Abbott Optical Adhesive Cover

Abbott Adhesive Cover Applicator

Sample Racks

Round-bottom 11.6 to 16 mm Sample Tubes

Vortex Mixer

Centrifuge capable of 2000 g

Calibrated Precision Pipettes capable of delivering 20 μL-1000 μL

20 μL-1000 μL Aerosol Barrier Pipette Tips for precision pipettes

Molecular Biology Grade Water (RNase Free)

1.7 mL Molecular Biology Grade Microcentrifuge Tubes (Dot Scientific, Inc. or equivalent)

Cotton Tip Applicators (Puritan or Equivalent)

NOTE: If required per the Contamination From External dU-Containing Amplified Product section of this package insert.

NOTE: These items are used in the procedure for Monitoring the Laboratory for the Presence of Amplification Product. Refer to the QUALITY CONTROL PROCEDURES section of this package insert. Abbott m2000rt Instrument

Abbott RealTime HCV Genotype II m2000 System Combined Application CD-ROM

Abbott m2000rt Optical Calibration Kit (List No. 04J71-93)

Other Materials

Biological safety cabinet approved for working with infectious materials.

Sealable plastic bags

Procedural Precautions

Read the instructions in this package insert carefully before processing samples.

The Abbott RealTime HCV Genotype II Internal Control, Negative Control, and Positive Control vials are intended for single-use only and should be discarded after use.

Sample tubes should be inspected for air bubbles. If found, remove them with a sterile pipette tip. Reagent bubbles may interfere with proper detection of reagent levels in the reagent vessel, causing insufficient reagent aspiration, which could impact results. Caution should be taken to avoid cross-contamination between samples by using a new sterile pipette tip for each tube.

Use aerosol barrier pipette tips or disposable pipettes only one time when pipetting specimens or Internal

Control. To prevent contamination to the pipette barrel while pipetting, care should be taken to avoid touching

the pipette barrel to the inside of the sample tube or container. The use of extended aerosol barrier pipette tips is recommended.

If the Abbott m2000sp master mix addition protocol is not initiated, re-cap the Amplification Reagent vials and return the Amplification Reagent Packs to –25 to –15°C storage. Once thawed, the Abbott RealTime HCV

Genotype II Amplification Reagent Packs can be frozen and thawed a maximum of three additional times. If the Abbott m2000sp master mix addition protocol is aborted, then discard the amplification reagents.

NOTE: The m2000rt protocol must be started within 90 minutes of the initiation of the Master Mix Addition protocol.

If the Abbott m2000rt instrument run is not initiated within 90 minutes, or is interrupted or aborted, seal the Abbott 96-Well Optical Reaction Plate in a sealable plastic bag and dispose according to the Abbott m2000rt Operations Manual along with the gloves used to handle the plate.

Monitoring procedures for the presence of amplification product can be found in the QUALITY CONTROL PROCEDURES section in this package insert.

To reduce the risk of nucleic acid contamination, clean and disinfect spills of specimens by including the use of a tuberculocidal disinfectant such as 1.0% sodium hypochlorite or other suitable disinfectant.

The Abbott RealTime HCV Genotype II Controls must be processed in conjunction with specimens to be tested. The use of the Abbott RealTime HCV Genotype II Controls is integral to the performance of the Abbott RealTime HCV Genotype II assay. Refer to the QUALITY CONTROL PROCEDURES section of this package insert for details.

ASSAY PROTOCOL

For a detailed description of how to operate the Abbott m2000sp instrument and Abbott m2000rt instrument, refer to the Abbott m2000sp and m2000rt Operations Manuals, Operating Instructions section.

Laboratory personnel must be trained to operate the Abbott m2000sp and m2000rt instruments. The operator must have a thorough knowledge of the applications run on the instruments and must follow good laboratory practices.

Sample Preparation Area

All specimen preparation must take place in the dedicated Sample Preparation Area. Refer to the Handling Precautions section of this package insert before preparing samples.

1. A maximum of 24 samples or 72 reactions (3 reactions per sample) can be performed per run. A negative control and a positive control are included in each run, therefore allowing a maximum of 22 specimens to be processed per run.

Check sample volume. The Abbott RealTime HCV Genotype II assay minimum sample volume and

associated rack requirements on the Abbott m2000sp are:

Rack Tube Diameter a

13 mm

16 mm

13mm

aMinimum Sample Volume 1.0 mL 1.3 mL 0.8 mL 11.6 to 14.0 mm 15.0 to 16.0 mm Reaction Vessel Refers to sample tube outer diameter

If frozen, thaw specimens at 15 to 30°C or at 2 to 8°C. Once thawed, specimens can be stored at

2 to 8°C for up to 6 hours before processing.

Before use, vortex specimens three times for 2 to 3 seconds. Ensure that bubbles or foam are not created. If found, remove them with a new sterile pipette tip for each tube. Specimens showing

particulate matter or turbidity should be clarified by centrifugation at 2000 g for 5 minutes prior

to testing. Aliquot each specimen into clean tubes or vials if necessary. Refer to the Abbott m2000sp Operations Manual for tube sizes. Avoid touching the inside of the cap when opening tubes.

2. Thaw assay controls and IC at 15 to 30°C or at 2 to 8°C; see QUALITY CONTROL PROCEDURES

section of this package insert.

Once thawed, assay controls and IC can be stored at 2 to 8°C for up to 24 hours before use.

Vortex each control three times for 2 to 3 seconds before use. Ensure that bubbles or foam are not created. If found, remove them with a new sterile pipette tip for each tube. Ensure that the contents of each vial are at the bottom after vortexing by tapping the vials on the bench to bring liquid to the bottom of the vial.

3. Thaw amplification reagents at 15 to 30°C or at 2 to 8°C and store at 2 to 8°C until required for the

amplification master mix procedure. This step can be initiated before completion of the sample preparation procedure.

Note: Do not vortex the Amplification Reagent Pack.

Once thawed, store at 2 to 8°C for up to 24 hours if amplification reagents are not being processed

immediately.

NOTE: Use one bottle of mLysis Buffer and two vials of Internal Control to support processing up to 24 samples. Each sample is tested with each of the three RealTime HCV Genotype II Amplification Reagent Packs. Use one each of RealTime HCV Genotype II Amplification Reagent Packs A, B, and C to support processing up to 24 samples.

Abbott m2000sp Procedure

4. Gently invert the Abbott mSample Preparation bottles to ensure a homogeneous solution. If crystals are observed in any of the reagent bottles upon opening, allow the reagent to equilibrate at room temperature until the crystals disappear. Do not use the reagents until the crystals have dissolved. Ensure bubbles or foam is not generated; if present, remove with a sterile pipette tip, using a new tip for each bottle.

5. Vortex each IC three times for 2 to 3 seconds before use. Ensure bubbles or foam is not generated; if present, remove with a sterile pipette tip, using a new tip for each vial.

6. Using a calibrated precision pipette DEDICATED FOR INTERNAL CONTROL USE ONLY, add 2000 μL of IC to one bottle of mLysis Buffer. Mix by gently inverting the container 5 to 10 times to minimize foaming.

7. Place the negative control, the positive control, and the patient specimens into the Abbott m2000sp sample rack.

8. Place the 5 mL Reaction Vessels into the m2000sp 1 mL subsystem carrier.

9. Load the carrier racks containing the Abbott mSample Preparation System reagents and the Abbott 96-Deep-Well Plate on the Abbott m2000sp worktable as described in the Abbott m2000sp Operations Manual, Operating Instructions.

10. From the Run Sample Extraction screen, select and initiate the sample extraction protocol as described in the m2000sp Operations Manual, Operating Instruction.

Following completion of the Sample Extraction protocol, proceed to Step 11 for master mix preparation or processed samples may be stored in the Abbott 96-Deep-Well Plate prior to initiating the Abbott m2000sp Master Mix Addition protocol:

At 2 to 30°C for up to 4 hours

At –20°C or colder for up to 7 days

Thaw processed samples prior to initiating the Master Mix Addition Protocol

NOTE: Change gloves before handling the amplification reagents.

11. Load the three Amplification Reagent Packs (A, B, and C) and three master mix vials on the m2000sp

worktable after sample preparation is completed. Load the three Amplification Reagent Packs (A, B, and C) into the reagent positions (1, 2, and 3), respectively.

The three RealTime HCV Genotype II Amplification Reagent Packs support up to 24 reactions.

Ensure the Amplification reagents are thoroughly thawed before use.

Label each vial cap within each amplification reagent pack (i.e. A, B, C) prior to opening the

amplification reagents.

Prior to opening the amplification reagents, ensure that the contents are at the bottom of the vials by tapping the vials in an upright position on the bench.

Remove the amplification vial caps.

12. Select the appropriate deep well plate from the Run Master Mix Addition screen that matches the

corresponding sample preparation extraction. Initiate the Abbott m2000sp Master Mix Addition protocol. Follow the instructions as described in the Abbott m2000sp Operations Manual, Operating Instructions section.

NOTE: The m2000rt protocol (step 17) must be started within 90 minutes of the initiation of the Master Mix Addition protocol (step 12).

If the Abbott m2000sp master mix addition protocol is not initiated, re-cap the Amplification Reagent vials and return the Amplification Reagent Packs to -25 to -15°C storage. Once thawed, the Abbott

RealTime HCV Genotype II Amplification Reagent Packs can be frozen and thawed a maximum of three additional times. If the Abbott m2000sp master mix addition protocol is aborted, then discard the

amplification reagents.

Amplification Area

13. Switch on and initialize the Abbott m2000rt in the amplification area.

The Abbott m2000rt requires 15 minutes to warm up.

NOTE: Change laboratory coats and gloves before returning to the sample preparation area.

14. Place the Abbott 96-Well Optical Reaction Plate into the Abbott Splash-Free Support Base after the Abbott m2000sp instrument has completed addition of samples and master mix.

15. Seal the Abbott 96-Well Optical Reaction Plate according to the Abbott m2000sp Operations Manual,

Operating Instructions section. Export the completed PCR plate results via a network connection directly to a mapped m2000rt (or indirectly via a CD).

Abbott m2000rt Procedures

For a detailed description of how to perform an Abbott m2000rt protocol, refer to the Operating Instructions section in the Abbott m2000rt Operations Manual.

16. Place the Abbott 96-Well Optical Reaction Plate in the Abbott m2000rt instrument. Import the m2000sp test order via a network connection directly to a mapped m2000rt (or indirectly via a CD) per the Import Order instructions in the Abbott m2000rt Operations Manual, Operating Instructions section.

17. Initiate the Abbott RealTime HCV Genotype II protocol as described in the Abbott m2000rt Operations Manual. The Abbott m2000rt completes the run in approximately 2 hours and forty-five minutes.

If the Abbott m2000rt instrument run is not initiated within 90 minutes from initiation of Master Mix

Addition (Step 12), or is interrupted or aborted, seal the Abbott 96-Well Optical Reaction Plate in a sealable plastic bag and dispose according to the Abbott m2000rt Operations Manual along with the gloves used to handle the plate.

18. After the Abbott m2000rt instrument has completed the amplification and detection protocol, remove the 96-Well Optical Reaction Plate and dispose according to the instructions in the Handling Precautions section of this package insert.

POST PROCESSING PROCEDURES

1. Remove the Abbott 96-Deep-Well Plate, Reaction Vessels, Reagent Vessels, Amplification Reagent Packs, and Master Mix Vials from the worktable and dispose of according to the Abbott m2000sp Operations Manual.

2. Place the Abbott 96-Well Optical Reaction Plate in a sealable plastic bag and dispose according to the Abbott m2000rt Operations Manual along with the gloves used to handle the plate.

3. Clean the Splash-Free Support Base before next use, according to the Abbott m2000rt Operations Manual. QUALITY CONTROL PROCEDURES

Abbott m2000rt Optical Calibration

Refer to the Calibration Procedures section in the Abbott m2000rt Operations Manual for a detailed description of how to perform an Abbott m2000rt Optical Calibration. Optical calibration of the Abbott m2000rt instrument is required for the accurate measurement and discrimination of dye fluorescence during the Abbott RealTime HCV Genotype II assay.

The following Abbott m2000rt Optical Calibration Plates are used to calibrate the Abbott m2000rt instrument for the Abbott RealTime HCV Genotype II assay:

FAM Plate (Carboxyfluorescein)

ROX Plate (Carboxy-X-rhodamine)

VIC® Plate (Proprietary dye)

NED Plate (Proprietary dye)

Cy5® Plate (Cyanine)

Detection of Inhibition

A defined, consistent quantity of IC nucleic acid is introduced into each specimen and control at the beginning of sample preparation and measured on the Abbott m2000rt to demonstrate proper specimen processing and assay validity. The IC is comprised of a RNA sequence unrelated to the HCV target sequences.

The median amplification cycle at which the IC target sequence fluorescent signal is detected in the negative and positive control samples establishes an IC validity range to be met by all subsequent processed specimens on that run.

An error is displayed when a specimen or control fails to meet their respective IC specification. Specimens whose IC CN value exceeds the established range must be retested starting with sample preparation. Refer to RESULTS section of this package insert and the Abbott m2000rt System Operations Manual for a list of error codes and flags.

Negative and Positive Controls

A negative control and a positive control ( are included in each test order to evaluate run validity. An error is displayed when a control result is invalid. Refer to the Abbott m2000rt Operations Manual for an explanation of the corrective actions for the error. If the negative or positive controls are invalid, all of the specimens and controls from that run must be reprocessed, beginning with sample preparation.

The presence of HCV must not be detected in the negative control. HCV detected in the negative control is indicative of contamination by other samples or by amplified product introduced during sample preparation or during preparation of the Abbott 96-Well Optical Reaction Plate. To avoid contamination, clean the Abbott m2000sp instrument and the Abbott m2000rt instrument according to the Operations Manuals and repeat the sample processing for controls and specimens following the Procedural Precautions section of this package insert. If negative controls are persistently reactive, contact Abbott Molecular Customer Service.

Monitoring the Laboratory for the Presence of Amplification Product

It is recommended that this test be done at least once a month to monitor laboratory surfaces and equipment for contamination by amplification product. It is very important to test all areas that may have been exposed to processed specimens, controls, and/or amplification product. This includes routinely handled objects such as pipettes, the Abbott m2000sp and Abbott m2000rt function keys, laboratory bench surfaces, and

microcentrifuges.

1. Add 0.8 mL RNase-free water to a separate 1.7 mL RNase-free microcentrifuge tube for each laboratory surface to be monitored.

2. Saturate the cotton tip of an applicator (Puritan or equivalent) in the RNase-free water from the

microcentrifuge tube.

3. Using the saturated cotton tip of the applicator, wipe the area to be monitored using a sweeping motion. Place the applicator back into the microcentrifuge tube from step 1.

4. Swirl the cotton tip in the RNase-free water 10 times, and then press the applicator along the inside of the microcentrifuge tube so that the liquid drains back into the solution at the bottom of the tube. Discard the applicator.

5. For each additional area to be monitored repeat steps 2 through 4.

Note: A small amount of mWash 1 buffer is added to each monitor sample in order to ensure that the ionic strength of the sample is sufficient for liquid level detection during processing on the m2000sp.

6. Pipette 0.5 mL of the mWash 1 buffer to a clean tube using the pipette dedicated for Internal Control use.

7. Add 20 μL of the mWash 1 buffer from step 6 to each microcentrifuge tube from step 4.

8. Cap the microcentrifuge tubes.

9. Transfer liquid from each microcentrifuge tube to unique 5 mL Reaction Vessels.

10. Bring the volume of each 5 mL Reaction Vessel to 1.5 mL with RNase-free water.

11. Place the 5 mL Reaction Vessels into the Abbott m2000sp sample rack and complete the assay following the ASSAY PROTOCOL section of this package insert.

The Uracil-N-Glycosylase (UNG) (List No. 08L21-66) protocol should not be used to monitor the

laboratory for presence of amplification product.

12. The presence of contamination is indicated by the detection of HCV nucleic acid in the swab samples.

13. If HCV nucleic acid is detected on equipment, follow the cleaning and decontaminating guidelines given in that equipment’s operations manual. If HCV nucleic acid is detected on surfaces, clean the contaminated areas with 1.0% (v/v) sodium hypochlorite solution, followed by 70% ethanol or water.

Note: Chlorine solutions may pit equipment and metal. Use sufficient amounts or repeated applications of 70% ethanol or water until chlorine residue is no longer visible.

14. Repeat testing of the contaminated area by following Steps 1 through 13.

RESULTS

Abbott RealTime HCV Genotype II is a qualitative assay. The Abbott RealTime HCV Genotype II Controls are used to establish run validity for the HCV Genotype II assay.

The Abbott RealTime HCV Genotype II assay employs two determinations on each assay response to accurately designate HCV genotypes:

Cycle Number (CN) and CN number difference, as compared to the HCV-All-probe cycle number, for each of the genotype

specific probes (1, 1a, 1b, 2, 3, 4, and 5).

A sample is reported to contain a HCV genotype when the CN threshold is exceeded, and when the genotype-specific CN value is within a predetermined number of cycles of the HCV-All CN value for the same specimen. Multiple genotypes can be detected simultaneously (see Interpretation of Results below).

INTERPRETATION OF RESULTS

If the controls are valid, then proceed to results and interpretations. The Abbott m2000rt instrument

automatically reports the genotype result on the Abbott m2000rt workstation. Assay results and interpretations will look similar to the following examples:

Location Sample ID Sample Type Result Interpretation

Flags Error Code A1 HCV-GT_NEG Control Passed B1 HCV-GT_POS Control Passed a C1 Patient1 1 1ab D1 Patient2 1 1bb

E1 Patient3 HCV not detectedc

F1 Patient4 2

G1 Patient5 2, 3d

H1 Patient6 HCVDetectede

a No Genotype Resultf The results log will indicate amplification curves for GT 1 and GT 4 based on the use of 5′ UTR RNA sequences. If the negative or positive controls are invalid, refer to Quality Control Procedures, Negative and Positive Control section of this package insert.

b Genotype 1a and genotype 1b results may be reported without genotype 1.

c The assay did not detect HCV. Ensure HCV concentration is at least 500 IU/mL and repeat RealTime HCV Genotype II assay. d When reporting multiple genotypes, consider inclusion of this statement: Multiple genotype assay results may be caused by a mixed genotype infection, recombination of HCV genotypes, or assay probe cross-reactivity. e,f The assay detected HCV but did not produce a genotype result. Ensure HCV concentration is at least 500 IU/mL and repeat RealTime HCV Genotype II assay. If repeat testing generates the same result, consider an alternative HCV genotype method.

For more information about error codes and flags, refer to the Abbott m2000rt Operations Manual.

LIMITATIONS OF THE PROCEDURE

FOR IN VITRO DIAGNOSTIC USE.

Optimal performance of this test requires appropriate specimen collection, handling, preparation, storage, and transport to the test site (refer to the SPECIMEN COLLECTION, STORAGE, AND

TRANSPORT TO THE TEST SITE section of this package insert).

Human serum or plasma (ACD-A, CPD, potassium EDTA, or sodium EDTA) specimens may be used with the Abbott RealTime HCV Genotype II assay. The use of other anticoagulants has not been

validated with the Abbott RealTime HCV Genotype II assay.

Use of the Abbott RealTime HCV Genotype II assay is limited to personnel who have been trained in the procedures of a molecular diagnostic assay and the Abbott m2000sp and m2000rt instruments.

The instruments and assay procedures reduce the risk of contamination by amplification product.

However, nucleic acid contamination from the positive control or specimens must be controlled by good laboratory practice and careful adherence to the procedures specified in this package insert.

A specimen with an interpretation of “No Genotype Result” can not be presumed to be negative for the tested genotypes.

Multiple genotype assay results may be caused by a mixed genotype infection, recombination of HCV genotypes, or assay probe cross-reactivity.17

The Abbott RealTime HCV Genotype II assay is capable of detecting both genotypes in a genotype mixture when the concentrations of both genotypes are near equal; however, the assay may not detect the lower concentration genotype.

Performance has not been established with the Abbott RealTime HCV Genotype II assay for HCV genotype 6 specimens.

HCV genotype 6 specimens may generate a HCV genotype 1 result with the Abbott RealTime HCV Genotype II assay based on probe cross-reactivity of the HCV genotype 1 probe.

As with any diagnostic test, results from the Abbott RealTime HCV Genotype II assay should be

interpreted in conjunction with other clinical and laboratory findings. A specimen with a result of “HCV not detected” cannot be presumed to be negative for HCV RNA.

Contamination from HCV positive controls and clinical specimens can be avoided only by good

laboratory practices and careful adherence to the procedures specified in this package insert.

SPECIFIC PERFORMANCE CHARACTERISTICS

REPRODUCIBILITY / PRECISION

The Reproducibility / Precision of Abbott RealTime HCV Genotype II was evaluated by testing a 36-member panel (2 vials of 18 unique members) representing HCV genotypes 1a, 1b, 2, 3, 4, and 5, each at three

concentration levels (500 to 1000, 5000 to 10000, > 50000 IU/mL). All panel members were composed of HCV positive donor units diluted in defibrinated human plasma.

A total of 3 Abbott RealTime HCV Genotype II Amplification reagent lots were used. Each of the 3 clinical sites tested 2 of the 3 Amplification reagent lots for 5 nonconsecutive days each, resulting in a total of 10 runs at each site.

The percent correctly identified rate for the Abbott RealTime HCV Genotype II assay was 99.8% (1070/1072) overall for genotypes 1 – 5. The overall No Result (“HCV detected, No Genotype Result” or “HCV not detected”) rate was 0.2% (2/1072) for genotypes 1 – 5.

Within-run, between-run, between-lot, between-site, and total standard deviations and %CV for cycle number (CN) were determined. The total SD ranged from 0.25 to 1.55, the within-run component SD ranged from 0.15 to 1.37, the between-run component SD ranged from 0.00 to 0.14, the between-lot component SD ranged from 0.00 to 0.73, and the between-site component SD ranged from 0.00 to 0.71.

The results, representative of the reproducibility / precision of the Abbott RealTime HCV Genotype II assay, are summarized in Tables 1 and 2.

Table 1. Abbott RealTime HCV Genotype II Reproducibility Study

Overall Analyses by HCV Genotype for all Sites and Lots Combined

Total Number of Number Percent Correct Detection Rate bb HCV Number of Correctly “No Result” 95% CI c Genotype Eligible Identified Determinations Percent Detected

Resultsa (T) Results (D) Panel NR (NR%) % [D/(T – NR)] LL UL

1a

1b

2

3

4

5

Overalld

a

b180 179 178 179 178 178 1072 180 179 178 179 177 177 1070 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 1 (0.6) 1 (0.6) 2 (0.2) 100 (180/180) 100 (179/179) 100 (178/178) 100 (179/179) 100 (177/177) 100 (177/177) 100 (1070/1070) 97.9 97.9 97.9 97.9 97.9 97.9 99.6 100 100 100 100 100 100 100 This number includes all Abbott RealTime HCV Genotype II valid assay results. For HCV genotypes 1 through 5, Abbott RealTime HCV Genotype II assay results “HCV Detected,

No Genotype Result” or “HCV not detected” are considered “No Result.” c 95% Lower and Upper Confidence Interval limits. d Denotes analysis based on assay results from all genotyped panels 1a, 1b, 2, 3, 4, and 5 combined.

Table 2. Abbott RealTime HCV Genotype II Precision Study – Overall Analysis all Sites and Lots Combined

HCV Panel Within-Run Between-Run Between-Lot Between-Site Genotype Concentration Mean Component Component Component Component Totald Panel Level n CN a SD %CV SD %CV SD %CV SD %CV SD %CV 1a High 60 22.95 0.24 1.1 0.14 0.6 0.08 0.3 0.14 0.6 0.32 1.4 1b High 59b 21.65 0.18 0.9 0.01 0.1 0.13 0.6 0.09 0.4 0.25 1.1

b2 High 58 21.81 0.25 1.2 0.00 0.0 0.16 0.7 0.12 0.5 0.32 1.5

3 High 60 20.87 0.47 2.2 0.00 0.0 0.34 1.6 0.00 0.0 0.58 2.8

b4 High 59 22.76 0.57 2.5 0.11 0.5 0.09 0.4 0.17 0.7 0.61 2.7

5 High 58b 21.40 0.41 1.9 0.00 0.0 0.43 2.0 0.71 3.3 0.92 4.3 1a Medium 60 25.29 0.27 1.1 0.00 0.0 0.11 0.4 0.11 0.4 0.31 1.2 1b Medium 60 24.45 0.15 0.6 0.00 0.0 0.10 0.4 0.17 0.7 0.25 1.0 2 Medium 60 23.77 0.26 1.1 0.00 0.0 0.18 0.8 0.16 0.7 0.36 1.5 3 Medium 60 24.15 0.39 1.6 0.00 0.0 0.30 1.3 0.00 0.0 0.49 2.0

b,c0.25 1.0 0.00 0.0 0.27 1.1 0.36 1.5 0.52 2.1 4 Medium 58 24.51

5 Medium 60 25.84 0.63 2.4 0.00 0.0 0.56 2.2 0.00 0.0 0.84 3.3 1a Low 60 29.11 0.27 0.9 0.12 0.4 0.13 0.4 0.08 0.3 0.33 1.1 1b Low 60 27.59 0.27 1.0 0.00 0.0 0.15 0.5 0.21 0.7 0.37 1.3 2 Low 60 25.94 1.37 5.3 0.00 0.0 0.73 2.8 0.00 0.0 1.55 6.0

b3 Low 59 27.67 1.00 3.6 0.00 0.0 0.00 0.0 0.36 1.3 1.06 3.8

4 Low 60 28.30 0.39 1.4 0.00 0.0 0.17 0.6 0.42 1.5 0.60 2.1

c5 Low 59 28.76 0.83 2.9 0.00 0.0 0.45 1.6 0.15 0.5 0.96 3.3

a

b HCV genotype probe specific cycle number. Invalid replicates (8 total) were not included in the analysis.

c Replicates without a HCV genotype identification (2 total) were not included in the analysis. d The total variability contains Within-Run, Between-Run, Between-Lot and Between-Site variability.

Limit of Detection (LoD) by Genotype

The assay limits of detection (LoD) were estimated for each HCV genotype (1a, 1b, 2, 3, 4, and 5). For each genotype, a single HCV specimen was diluted in HCV negative human plasma as well as in human serum to make panels containing the following HCV concentrations: 1000, 500, 250, 100, and 25 IU/mL.

Each panel member was tested with a minimum of 2 replicates per run, with 2 runs per day, for 4 or 5 days and with 2 Abbott RealTime HCV Genotype II Amplification Reagent lots, for a total of 40 measurements. The results for each HCV genotype in plasma and serum are summarized in Table 3.

Table 3. Abbott RealTime HCV Genotype II - Limit of Detection Summary Limit of Detection Estimates (IU/mL) Sample Type

Plasma

Serum

HCV Genotype 1a 1b 2 3 4 5 100 100 500 500 500 250 100 25 500 500 100 100

The limit of detection of the Abbott RealTime HCV Genotype II assay is 500 IU/mL.

Accuracy

The accuracy of the Abbott RealTime HCV Genotype II assay was evaluated by testing 266 HCV genotype 1 (144 of genotype 1a, 122 of genotype 1b), 116 HCV genotype 2, 87 HCV genotype 3, 79 HCV genotype 4, and 27 HCV genotype 5 specimens. Nucleotide sequencing was used to determine the reference genotype of each specimen tested in this study.

The percent correctly identified (Accuracy) rate for the Abbott RealTime HCV Genotype II assay while

excluding “No Result” determinations was 99.6% (265/266) for HCV genotype 1, 99.1% (110/111) for HCV genotype 2, 100.0% (86/86) for HCV genotype 3, 98.7% (76/77) for HCV genotype 4, and 100.0% (24/24) for HCV genotype 5, and 99.3% (139/140) for subtype 1a and 99.1% (114/115) for subtype 1b.

The percent correctly identified (Accuracy) rate for the Abbott RealTime HCV Genotype II assay while excluding “No Result” determinations was 99.5% (561/564) overall for genotypes 1 – 5.

The results, representative of the Abbott RealTime HCV Genotype II assay, are summarized in Tables 4 and 5.

Table 4. Abbott RealTime HCV Genotype II– Accuracy Analysis for HCV Genotypes 1 Through 5 HCV

Genotype

By

Sequencing Total Number of Number of Eligible ResultsEligible Excluding aResults "No Result"b Number of RealTime Results in Agreement with Sequencing Percent Correctly Identified (Accuracy) c LL UL

1d 266 266 265 99.6 (265/266) 97.9 99.9 2e 116 111 110 99.1 (110/111) 95.1 99.8

86 86 100 (86/86) 95.7 100 3f 87

77 76 98.7 (76/77) 93.0 99.8 4g 79

24 24 100 (24/24) 86.2 100 5h 27

Overall

a 1 through 5 i575 564 561 99.5(561/564) 98.4 99.8 This number includes all valid Abbott RealTime HCV Genotype II assay and Sequencing results and excludes

11 mixed infection results by the Abbott RealTime HCV Genotype II assay. b For HCV genotypes 1 through 5, Abbott RealTime HCV Genotype II assay results “HCV Detected, No Genotype Result” or “HCV not detected” are considered “No Result.” c 95% Lower and Upper Confidence Interval limits. d Includes HCV genotype 1 and all subtypes of 1 (1a, 1b) results. e Four out of 116 HCV genotype 2 samples were identified as “HCV Detected, No Genotype Result” and one sample was

identified as “HCV not detected.” f One out of 87 HCV genotype 3 samples was identified as “HCV Detected, No Genotype Result.” g Two out of 79 HCV genotype 4 samples were identified as “HCV Detected, No Genotype Result.” h Two out of 27 HCV genotype 5 samples were identified as “HCV Detected, No Genotype Result” and one sample was

identified as “HCV not detected.” I Denotes analysis based on results from HCV genotypes 1 through 5 combined.

Table 5. Abbott RealTime HCV Genotype II– Accuracy Analysis for HCV Genotypes 1a and 1b Number of Number of HCV Total

Genotype Number of Eligible ResultsRealTime Results

Excluding in Agreement By Eligible abSequencingResults "No Result" with Sequencing

1a

1b

a Percent Correctly Identified (Accuracy) LL UL cd144 140 122 115 139 99.3 (139/140) 96.1 99.9 114 99.1 (114/115) 95.2 99.8 eThis number includes all valid Abbott RealTime HCV Genotype II assay and Sequencing results and excludes

4 mixed infection results by the Abbott RealTime HCV Genotype II assay. b For HCV subtypes 1a and 1b, Abbott RealTime HCV Genotype II Assay results “HCV Detected, No Genotype Result,”

“HCV not detected,”or HCV genotype “1” without being subtyped are considered “No Result.” c 95% Lower and Upper Confidence Interval limits. d Four out of 144 HCV genotype 1a samples were identified as genotype 1 only but not as subtype 1a. e Seven out of 122 HCV genotype 1b samples were identified as genotype 1 but not as subtype 1b.

The overall Abbott RealTime HCV Genotype II “No Result” rate for all HCV genotypes combined was 1.9% (11/575). Overall, HCV genotype 1 samples were not subtyped in 4.1% (11/266) of specimens: 2.8% (4/144) for subtype 1a and 5.7% (7/122) for subtype 1b.

Analytical Specificity

Potentially Interfering Substance

The susceptibility of the Abbott RealTime HCV Genotype II assay to interference by elevated levels of potentially interfering substances was evaluated in two studies.

In the first study, HCV negative plasma samples and plasma samples containing 10,000 IU/mL of HCV genotype 2 armored RNA were spiked with high levels of hemoglobin (500 mg/dL), bilirubin (20 mg/dL), protein (9 g/dL), or triglycerides (3000 mg/dL) and tested. In the second study, HCV negative plasma samples and plasma samples containing 1000 IU/mL of HCV genotype 1 virus were spiked with high levels of hemoglobin (2 g/L), bilirubin (342 uM), protein (120 g/L), or triglycerides (37.34 mM) and tested.

No interference in the performance of the Abbott RealTime HCV Genotype II assay was observed in the presence of the endogenous substances for all HCV positive and negative samples tested.

Two studies were conducted to assess the susceptibility of the assay to potential interference from high levels of drugs commonly prescribed for the treatment of hepatitis C virus (HCV) and other related diseases. Antivirals and antibiotics at concentrations in excess of peak plasma or serum levels were tested. The drugs listed below were tested in five pools in which each drug was present in excess of reported peak plasma or serum levels.

Each drug pool was spiked into an HCV serologically negative plasma aliquot and HCV positive plasma aliquot (HCV genotype 2 armored RNA at 10,000 IU/mL in Study 1 and HCV genotype 1 virion at 1000 IU/mL in Study 2) for testing. Non-spiked aliquots were used as controls. No interference in the performance of the Abbott RealTime HCV Genotype II assay was observed in the presence of the following drugs for all HCV positive and negative samples tested:

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