Abbott RealTime

HIV-1 en
02G31-010
02G31-010 51-608282/R9
51-608282/R9
NOTE: CHANGES HIGHLIGHTED
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Reference Number This package insert must be read carefully prior to use. Package insert
instructions must be followed accordingly. Reliability of assay results
Lot Number cannot be guaranteed if there are any deviations from the instructions in
this package insert.
In Vitro Diagnostic Medical Device
NAME
In Vitro Test Abbott RealTime HIV-1
INTENDED USE
Use By The Abbott RealTime HIV-1 assay is an in vitro reverse transcription
polymerase chain reaction (RT-PCR) assay for the quantitation of
Human Immunodeficiency Virus type 1 (HIV-1) in whole blood spotted
Contains sufficient for <n> tests on cards as dried blood spots (DBS) (i.e. obtained via venipuncture
or capillary blood) or human plasma from HIV-1 infected individuals.
Negative Control The Abbott RealTime HIV-1 is intended for use in conjunction with
clinical presentation and other laboratory markers as an indicator of
disease prognosis and for use as an aid in assessing viral response to
Low Positive Control
antiretroviral treatment as measured by changes in DBS or plasma HIV-1
RNA levels. This assay is not intended to be used as a screening test for
High Positive Control HIV-1 or as a diagnostic test to confirm the presence of HIV-1 infection.
INTENDED USER
Calibrator A
The intended users for the Abbott RealTime HIV-1 assay are laboratory
and healthcare professionals.
Calibrator B
SUMMARY AND EXPLANATION OF THE TEST
Human Immunodeficiency Virus (HIV) is the etiologic agent of Acquired
Immunodeficiency Syndrome (AIDS).1-3 It can be transmitted through
Internal Control sexual contact, exposure to infected blood or blood products, or from
an infected mother to the fetus.4 Acute HIV syndrome, characterized
by flu-like symptoms, develops 3 to 5 weeks after initial infection and
Amplification Reagent Pack is associated with high levels of viremia.5,6 Within 4 to 6 weeks of the
onset of symptoms, HIV specific immune response is detectable.7,8 After
Maximum Time Allowed seroconversion, viral load in peripheral blood declines and most patients
enter an asymptomatic phase that can last for years.9
Quantitative measurement of HIV levels in peripheral blood has
Upper Limit of Temperature greatly contributed to the understanding of the pathogenesis of HIV
infection10,11 and has been shown to be an essential parameter in
prognosis and management of HIV infected individuals.12-17 Decisions
Consult instructions for use regarding initiation or changes in antiretroviral therapy are guided by
monitoring plasma or DBS HIV RNA levels (viral load), CD4+ T cell
count, and the patient’s clinical condition.17-19 The goal of antiretroviral
Caution therapy is to reduce the HIV virus in plasma and DBS to below
detectable levels of available viral load tests.17,20
HIV RNA levels in plasma or DBS can be quantitated by nucleic acid
amplification or signal amplification technologies.19-23 The Abbott
Warning RealTime HIV-1 assay uses Polymerase Chain Reaction (PCR)
technology with homogenous real-time fluorescent detection. Partially
double-stranded fluorescent probe design allows detection of diverse
Authorized Representative in the group M subtypes and group O isolates. The assay is standardized
European Community against a viral standard from the Virology Quality Assurance (VQA)
Laboratory of the AIDS Clinical Trial Group,24 and against World
Manufacturer Health Organization (WHO) 1st International Standard for HIV-1 RNA
(97/656).25,26 The assay results can be reported in copies/mL or
See REAGENTS section for a full explanation of symbols used in International Units/mL (IU/mL) or their log base 10 equivalents.
reagent component naming. BIOLOGICAL PRINCIPLES OF THE PROCEDURE
The Abbott RealTime HIV-1 assay consists of 3 reagent kits:
NOTICE TO USER
• Abbott RealTime HIV-1 Amplification Reagent Kit
If a serious incident occurs in relation to this device, the incident should
• Abbott RealTime HIV-1 Control Kit
be reported to the manufacturer and to the appropriate competent
authority of the member state in which the user and/or the patient is • Abbott RealTime HIV-1 Calibrator Kit
established. To report to the manufacturer, see the contact information The Abbott RealTime HIV-1 assay uses RT-PCR27 to generate amplified
provided in the Customer service section or Technical assistance product from the RNA genome of HIV-1 in clinical specimens. An RNA
section of these instructions. sequence that is unrelated to the HIV-1 target sequence is introduced

1
into each specimen at the beginning of sample preparation. This probe and has a quencher molecule at its 3´ end. In the absence
unrelated RNA sequence is simultaneously amplified by RT-PCR, and of HIV‑1 target, the HIV-1 probe fluorescence is quenched through
serves as an internal control (IC) to demonstrate that the process hybridization to the quencher oligonucleotide. In the presence of the
has proceeded correctly for each sample. The amount of HIV‑1 target HIV‑1 target sequence, the HIV-1 probe preferentially hybridizes to
sequence that is present at each amplification cycle is measured the target sequence, dissociating from the quencher oligonucleotide,
through the use of fluorescent-labeled oligonucleotide probes on the allowing fluorescent detection.
Abbott m2000rt instrument. The probes do not generate signal unless The IC probe is a single-stranded DNA oligonucleotide with a fluorophore
they are specifically bound to the amplified product. The amplification at the 5´ end and a quencher at the 3´ end. In the absence of IC target
cycle at which fluorescent signal is detected by the Abbott m2000rt is sequences, probe fluorescence is quenched. In the presence of IC
proportional to the log of the HIV-1 RNA concentration present in the target sequences, probe hybridization to complementary sequences
original sample. separates the fluorophore and the quencher and allows fluorescent
Sample Preparation emission and detection.
The purpose of sample preparation is to extract and concentrate the The HIV-1 and IC specific probes are each labeled with a different
target RNA molecules to make the target accessible for amplification, fluorophore, thus allowing for simultaneous detection of both amplified
and to remove potential inhibitors of amplification from the extract. products at each cycle. The amplification cycle at which fluorescent
The Abbott mSample Preparation System (4 × 24 Preps) uses magnetic signal is detected by the Abbott m2000rt is proportional to the log of the
particle technology to capture nucleic acids and washes the particles HIV-1 RNA concentration present in the original sample.
to remove unbound sample components. The bound nucleic acids are Optional Amplification Reagent Extended Use Feature
eluted and transferred to output tubes or a 96 deep-well plate. The An overview of this feature is provided in Appendix 1 of this package
nucleic acids are then ready for amplification. The IC is taken through insert.
the entire sample preparation procedure along with the calibrators,
controls, and specimens. The optional amplification reagent extended use feature allows
amplification reagent packs containing prepared master mix to be stored
Two automated instrument systems, the Abbott m2000sp or the Abbott at – 25 to – 15°C, capped and protected from light, for up to 7 days
m1000 System can be used to prepare samples for the Abbott RealTime before a second use. The internal control (IC) may be used again within
HIV-1 assay. The Abbott m2000sp provides automated sample eluate 14 days if the vial remains capped at – 25 to – 15°C until the second
transfer and reaction assembly in the Abbott 96-Well Optical Reaction use. The amplification reagent extended use feature applies only to
Plate, while the Abbott m1000 System requires manual sample eluate samples prepared using the m2000sp system. Amplification reagent
transfer and reaction assembly. packs and IC can be used a total of 2 times. Throughout this manual,
Alternatively, samples can be prepared manually using the Abbott amplification reagent packs and IC that have not yet been used will be
mSample Preparation System, followed by manual reaction assembly. referred to as new amplification reagent packs and IC (ie, initial use).
Reagent Preparation and Reaction Plate Assembly Amplification reagent packs that have been used once and contain
The Abbott m2000sp combines the Abbott RealTime HIV-1 amplification prepared master mix will be referred to as partial amplification reagent
reagent components (HIV‑1 Oligonucleotide Reagent, Thermostable rTth packs. IC vials that have been used once will be referred to as partial
Polymerase Enzyme, and Activation Reagent). The Abbott m2000sp vials of IC.
dispenses the resulting master mix to the Abbott 96-Well Optical PREVENTION OF NUCLEIC ACID CONTAMINATION
Reaction Plate along with aliquots of the nucleic acid samples prepared The possibility of nucleic acid contamination is minimized because:
by the Abbott m2000sp. The plate is ready, after manual application of
the optical seal, for transfer to the Abbott m2000rt. • Reverse transcription, PCR amplification, and oligonucleotide
hybridization occur in a sealed Abbott 96-Well Optical Reaction
Abbott m1000 System users and manual sample preparation method Plate.
users manually combine the Abbott RealTime HIV-1 amplification reagent
components to create the amplification master mix and transfer aliquots • Detection is carried out automatically without the need to open the
of the master mix and sample eluates to the reaction plate. The plate is Abbott 96-Well Optical Reaction Plate.
ready, after manual application of the optical seal and centrifugation, for • Pipettes with aerosol barrier tips or disposable transfer pipettes are
transfer to the Abbott m2000rt. used for all pipetting. The disposable pipettes or pipette tips are
discarded after use.
Amplification
• Separate, dedicated areas are used to perform the Abbott RealTime
During the amplification reaction on the Abbott m2000rt, the target HIV-1 assay. Refer to the SPECIAL PRECAUTIONS section of this
RNA is converted to cDNA by the reverse transcriptase activity of the package insert.
thermostable rTth DNA polymerase. First, the HIV-1 and IC reverse
primers anneal to their respective targets and are extended during a REAGENTS
prolonged incubation period. After a denaturation step, in which the Abbott RealTime HIV-1 Amplification Reagent Kit
temperature of the reaction is raised above the melting point of the (List No. 02G31-010)
double-stranded cDNA:RNA product, a second primer anneals to the 1. Abbott RealTime HIV-1 Internal Control (List
cDNA strand and is extended by the DNA polymerase activity of the rTth No. 2G31Y0002) (4 vials, 1.2 mL per vial)
enzyme to create a double-stranded DNA product.
• < 0.01% noninfectious Armored RNA with internal control
During each round of thermal cycling, amplification products dissociate sequences in negative human plasma. Negative human plasma
to single strands at high temperature allowing primer annealing and tested and found to be nonreactive for HBsAg, HIV RNA, HCV
extension as the temperature is lowered. Exponential amplification of RNA, HBV DNA, anti‑HIV‑1/HIV‑2, and anti-HCV. Preservatives:
the product is achieved through repeated cycling between high and low 0.1% ProClin® 300 and 0.15% ProClin 950.
temperatures, resulting in a billion-fold or greater amplification of target
sequences. Amplification of both targets (HIV-1 and IC) takes place 2. Abbott RealTime HIV-1
simultaneously in the same reaction. Amplification Reagent Pack (List No. 2G31)
(4 packs, 24 tests/pack)
The target sequence for the Abbott RealTime HIV-1 assay is in the pol
integrase region of the HIV-1 genome. This region is highly conserved.28 • 1 bottle (0.141 mL) Thermostable rTth Polymerase Enzyme (2.9 to
The primers are designed to hybridize to the pol integrase region with 3.5 Units/µL) in buffered solution.
the fewest possible mismatches among various subtypes. • 1 bottle (1.10 mL) HIV-1 Oligonucleotide Reagent. < 0.1%
The IC target sequence is derived from the hydroxypyruvate reductase synthetic oligonucleotides (4 primers, 2 probes, and 1 quencher
gene from the pumpkin plant, Cucurbita pepo, and is delivered in an oligonucleotide), and < 0.3% dNTPs in a buffered solution with a
Armored RNA® particle that has been diluted in negative human plasma. reference dye. Preservatives: 0.1% ProClin 300 and 0.15%
ProClin 950.
Detection • 1 bottle (0.40 mL) Activation Reagent. 30 mM manganese
During the read cycles of amplification on the Abbott m2000rt, the chloride solution.
temperature is lowered further to allow fluorescent detection of Preservatives: 0.1% ProClin 300 and 0.15% ProClin 950.
amplification products as the HIV-1 and IC probes anneal to their targets NOTE: To use the amplification reagent extended use feature, reagent
(real-time fluorescence detection). The HIV-1 probe has a fluorescent packs with a 6-digit serial number above the barcodes must
moiety that is covalently linked to the 5´ end. A short oligonucleotide be used.
(quencher oligonucleotide) is complementary to the 5´ end of the HIV‑1

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Abbott RealTime HIV-1 Control Kit (List No. 2G31-80) (EC no. 247-500-7) and 2-methyl-2H-isothiazol-3-one
1. Abbott RealTime HIV-1 Negative Control (EC no. 220-239-6)(3:1)
(List No. 2G31Z) (8 vials, 1.8 mL per vial) Negative human plasma • Reaction mass of: 5-chloro-2-methyl-4-isothiazolin-3-one
tested and found to be nonreactive for HBsAg, HIV RNA, HCV RNA, (EC no. 247-500-7) and 2-methyl-4-isothiazolin-3-one
HBV DNA, anti-HIV-1/HIV-2, and anti-HCV. (EC no. 220-239-6)(3:1)
Preservatives: 0.1% ProClin 300 and 0.15% ProClin 950. The following warnings apply:
2. Abbott RealTime HIV-1 Low Positive Control Warning
(List No. 2G31W) (8 vials, 1.8 mL per vial) Noninfectious Armored
H317 May cause an allergic skin reaction.
RNA with HIV-1 sequences in negative human plasma. Negative
human plasma tested and found to be nonreactive for HBsAg, P261 Avoid breathing mist/vapours/spray.
HIV RNA, HCV RNA, HBV DNA, anti‑HIV‑1/HIV‑2, and anti-HCV. P272 Contaminated work clothing should not be
Preservatives: 0.1% ProClin 300 and 0.15% ProClin 950. allowed out of the workplace.
3. Abbott RealTime HIV-1 High Positive Control P280 Wear protective gloves/protective clothing/
(List No. 2G31X) (8 vials, 1.8 mL per vial). Noninfectious Armored eye protection.
RNA with HIV-1 sequences in negative human plasma. Negative P302+P352 IF ON SKIN: Wash with plenty of water.
human plasma tested and found to be nonreactive for HBsAg, P333+P313 If skin irritation or rash occurs: Get medical
HIV RNA, HCV RNA, HBV DNA, anti‑HIV‑1/HIV‑2, and anti-HCV. advice/attention.
Preservatives: 0.1% ProClin 300 and 0.15% ProClin 950.
P362+P364 Take off contaminated clothing and wash
Abbott RealTime HIV-1 Calibrator Kit (List No. 2G31-70) before reuse.
1. Abbott RealTime HIV-1 Calibrator A (List No. 2G31A) P501 Dispose of contents/container in
(12 vials, 1.8 mL per vial). Noninfectious Armored RNA with HIV-1 accordance with local regulations.
sequences in negative human plasma. Negative human plasma
tested and found to be nonreactive for HBsAg, HIV RNA, HCV RNA, SPECIAL PRECAUTIONS
HBV DNA, anti-HIV-1/HIV-2, and anti-HCV. Handling Precautions for Plasma Specimens
Preservatives: 0.1% ProClin 300 and 0.15% ProClin 950. The Abbott RealTime HIV-1 assay is only for use with plasma specimens
2. Abbott RealTime HIV-1 Calibrator B (List No. 2G31B) that have been handled and stored in capped tubes as described in the
(12 vials, 1.8 mL per vial). Noninfectious Armored RNA with HIV-1 SPECIMEN COLLECTION, STORAGE, AND TRANSPORT TO THE TEST
sequences in negative human plasma. Negative human plasma SITE section.
tested and found to be nonreactive for HBsAg, HIV RNA, HCV RNA,
HBV DNA, anti-HIV-1/HIV-2, and anti-HCV.
Handling Precautions for DBS Specimens
Preservatives: 0.1% ProClin 300 and 0.15% ProClin 950. The Abbott RealTime HIV-1 assay is only for use with whole blood or
DBS specimens that have been handled and stored as described in the
WARNINGS AND PRECAUTIONS SPECIMEN COLLECTION, STORAGE, AND TRANSPORT TO THE TEST
SITE section.
For In Vitro Diagnostic Use During preparation of samples, compliance with good laboratory
This assay is not intended to be used as a screening test for HIV-1 or as practices is essential to minimize the risk of cross-contamination
a diagnostic test to confirm the presence of HIV-1 infection. between samples and the inadvertent introduction of ribonucleases
(RNases) into samples during and after the extraction procedure. Proper
Safety Precautions
aseptic technique should always be used when working with RNA.
Refer to the Abbott m1000 Operating Manual, Safety Section, the
Amplification technologies such as PCR are sensitive to accidental
Manual Sample Preparation for Abbott RealTime RNA Assays Procedure,
introduction of product from previous amplification reactions. Incorrect
Handling Precaution Section, or Abbott m2000sp and Abbott m2000rt
results could occur if either the clinical specimen or the reagents
Operations Manuals, Hazard Section, for instructions on safety
used become contaminated by accidental introduction of even a
precautions.
few molecules of amplification product. Measures to reduce the risk
of contamination in the laboratory include physically separating the
CAUTION: This preparation contains human sourced and/or activities involved in performing PCR in compliance with good laboratory
potentially infectious components. Components sourced from human practices.
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 Work Areas
HBsAg. The material is also tested and found to be negative by FDA- Use 3 dedicated areas within the laboratory for performing the Abbott
licensed PCR methods for HIV-1 RNA and HCV RNA. No known test RealTime HIV-1 assay with the Abbott m1000 System or manual
method can offer complete assurance that products derived from human sample preparation using the Abbott mSample Preparation System
sources or inactivated microorganisms will not transmit infection. These and Abbott m2000rt:
reagents and human specimens should be handled as if infectious • The Reagent Preparation Area is dedicated to combining the Abbott
using laboratory safety procedures, such as those outlined in Biosafety RealTime HIV-1 amplification reagent components to create the
in Microbiological and Biomedical Laboratories,29 OSHA Standards amplification master mix and transferring aliquots of the master mix
on Bloodborne Pathogens,30 CLSI Document M29-A3,31 and other to the reaction plate. Laboratory coats, pipettes, pipette tips, and
appropriate biosafety practices.32 Therefore all human sourced materials vortexers used in the Reagent Preparation Area must remain in this
should be considered infectious. area and not be moved to either the Sample Preparation Area or
These precautions include, but are not limited to, the following: the Amplification Area.
• Wear gloves when handling specimens or reagents. • The Sample Preparation Area is dedicated to processing samples
• Do not pipette by mouth. (specimens, Abbott RealTime HIV‑1 Controls, and Calibrators),
and to adding processed samples, controls, and calibrators to the
• Do not eat, drink, smoke, apply cosmetics, or handle contact lenses
Abbott 96-Well Optical Reaction Plate. All reagents used in the
in areas where these materials are handled.
Sample Preparation Area should remain in this dedicated area at
• Clean and disinfect spills of specimens by including the use of a all times. Laboratory coats, pipettes, pipette tips, and vortexers
tuberculocidal disinfectant such as 1.0% sodium hypochlorite or used in the Sample Preparation Area must remain in this area
other suitable disinfectant.29 and not be moved to either the Reagent Preparation Area or the
• Decontaminate and dispose of all potentially infectious materials in Amplification Area. Do not bring amplification product into the
accordance with local, state, and federal regulations.32 Sample Preparation Area.
Components of the Abbott RealTime HIV-1 Calibrator Kit (2G31-70) • The Amplification Area is dedicated to the amplification and
and Abbott RealTime HIV-1 Control Kit (2G31-80), and the HIV-1 detection of amplified product. Laboratory coats and equipment
Oligonucleotide Reagent, HIV-1 Internal Control and Activation Reagent used in the Amplification Area must remain in this area and not
contain the following components: be moved to either the Reagent Preparation Area or the Sample
• 2-Methyl-2H-isothiazol-3-one (EC no. 220-239-6) Preparation Area.
• Reaction mass of: 5-chloro-2-methyl-4-isothiazolin-3-one

3
Only 2 dedicated areas, Sample Preparation Area and Amplification Contamination From External dU-Containing
Area, are recommended when the Abbott m2000sp and Abbott Amplified Product
m2000rt are used. Laboratories that use or have used HIV-1 amplification assays
Components contained within a kit are intended to be used together. that include post-PCR processing of the amplified product may be
Do not mix components from different kit lots. For example, do not use contaminated by dU-containing amplified product. Such contamination
the negative control from control kit lot X with the positive controls from may cause inaccurate results in the Abbott RealTime HIV-1 assay. Refer
control kit lot Y. to the Monitoring the Laboratory for the Presence of Contamination
Do not use kits or reagents after the expiration dates shown on kit section of the package insert. When negative controls are persistently
labels. reactive or where contamination with dU-containing HIV-1 amplified
Work area and instrument platforms must be considered potential product is likely to have occurred, it is recommended that the laboratory
sources of contamination. Change gloves after contact with potential use the uracil-N‑glycosylase (UNG) (List No. 06L87-02) contamination
contaminants (specimens, eluates, and/or amplified product) before control procedure if decontamination of the laboratory is unsuccessful.
handling unopened reagents, negative control, positive controls, Refer to Appendix 2 for the optional UNG Procedure.
calibrators, or specimens. Refer to the Abbott m2000sp and Abbott STORAGE INSTRUCTIONS
m2000rt Operations Manuals for instrument cleaning procedures.
Abbott RealTime HIV-1 Amplification Reagent Kit (List No. 02G31-010)
If the Abbott m1000 System or Abbott m2000sp instrument run is
aborted, dispose of all commodities and reagents according to the -15°C • New and Partial Abbott RealTime HIV-1 Amplification
Abbott m1000 Operating Manual or the Abbott m2000sp Operations Reagent Packs and Internal Control (IC) vials must
-25°C
Manual. be stored at – 25 to – 15°C when not in use. Care
NOTE: New amplification reagents may be saved, stored, and used a must be taken to separate the Abbott RealTime
second time, as described in this manual. HIV-1 Amplification Reagent Pack that is in use from
direct contact with samples, calibrators, and controls.
If the Abbott m2000sp master mix addition protocol is aborted, seal
the Abbott 96-Well Optical Reaction Plate in a sealable plastic bag and • Partial amplification reagent packs and IC must
dispose according to the Abbott m2000sp Operations Manual, Hazards be stored at – 25 to – 15°C, capped, upright, and
section, along with the gloves used to handle the plate. protected from light, following initial use.
If stored this way, partial amplification reagent packs
If the Abbott m2000rt instrument run is interrupted or aborted, seal the
with prepared master mix may be used a second
Abbott 96-Well Optical Reaction Plate in a sealable plastic bag and
time within 7 days of initial use. IC may also be used
dispose according to the Abbott m2000rt Operations Manual along with
a second time within 14 days of being thawed, if
the gloves used to handle the plate.
stored capped at – 25 to – 15°C.
Decontaminate and dispose of all potentially biohazardous materials
• After 2 uses, discard partial amplification reagent
in accordance with local, state, and federal regulations.31 All materials
packs and IC.
should be handled in a manner that minimizes the chance of potential
contamination of the work area. Abbott RealTime HIV-1 Control Kit (List No. 2G31-80)
NOTE: Autoclaving the sealed Reaction Plate will not degrade the • The Abbott RealTime HIV-1 Negative and Positive
amplified product and may contribute to the release of the Controls must be stored at – 10°C or colder.
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. Abbott RealTime HIV-1 Calibrator Kit (List No. 2G31-70)
Aerosol Containment • The Abbott RealTime HIV-1 Calibrator A and
To reduce the risk of nucleic acid contamination due to aerosols formed Calibrator B must be stored at – 10°C or colder.
during manual pipetting, aerosol barrier pipette tips must be used for
all manual pipetting. The pipette tips must be used only 1 time. Clean
and disinfect spills of specimens and reagents as stated in the Abbott SHIPPING CONDITIONS
m1000 Operating Manual or the Abbott m2000sp and Abbott m2000rt • Abbott RealTime HIV-1 Amplification Reagent Kit: Ship on dry ice.
Operations Manuals. • Abbott RealTime HIV-1 Control Kit: Ship on dry ice.
Contamination and Inhibition • Abbott RealTime HIV-1 Calibrator Kit: Ship on dry ice.
The following precautions should be observed to minimize the risks INDICATION OF INSTABILITY OR DETERIORATION OF
of RNase contamination, cross-contamination between samples, and REAGENTS
inhibition: When a positive or negative control value is out of the expected range,
• Wear appropriate personal protective equipment at all times. it may indicate deterioration of the reagents. Associated test results
• Use powder-free gloves. are invalid and samples must be retested. Assay recalibration may be
• Change gloves after having contact with potential contaminants necessary.
(such as specimens, eluates, and/or amplified product). INSTRUMENT PROCEDURE
• To reduce the risk of nucleic acid contamination due to aerosols The nucleic acid testing (NAT) software must be installed on the Abbott
formed during pipetting, pipettes with aerosol barrier tips must be m1000 System prior to performing the assay. For detailed information on
used for all pipetting. The length of the tip should be sufficient to NAT software installation, refer to the Abbott m1000 Operating Manual,
prevent contamination of the pipette barrel. While pipetting, care Putting into Operation section.
should be taken to avoid touching the pipette barrel to the inside of
the sample tube or container. The use of extended aerosol barrier The Abbott RealTime HIV-1 application files with the extended use
pipette tips is recommended. feature enabled must be installed on the Abbott m2000sp and Abbott
m2000rt systems from the Abbott RealTime HIV-1 m2000 ROW System
• Change aerosol barrier pipette tips between ALL manual liquid
Combined Application CD-ROM (List No. 1L68-014 or higher) prior
transfers.
to performing the assay. For detailed information on application file
• The Abbott mSample Preparation System (4 × 24 Preps) reagents installation, refer to the Abbott m2000sp and Abbott m2000rt Operations
are single use only. Use new reagent troughs or vessels, reaction Manuals, Operating Instructions section.
vessels, and newly opened reagents for every new Abbott RealTime
HIV‑1 assay run. At the end of each run, discard all remaining
reagents from the worktable as stated in the Abbott m1000
Operating Manual or the Abbott m2000sp Operations Manual and
the Abbott mSample Preparation System (4 × 24 Preps) product
information sheet.
• Follow instructions in this manual to recap and store amplification
reagents that are to be used a second time.

4
SPECIMEN COLLECTION, STORAGE, AND TRANSPORT For Abbott m1000 System For Abbott m2000sp Instrument
TO THE TEST SITE Sample Preparation Area Sample Preparation Area
Specimen Collection and Storage • Abbott m1000 System • Abbott m2000sp with software
Freshly drawn whole blood (ACD-A and EDTA) may be held at 15 version 6.0 or higher
to 30°C for up to 24 hours or at 2 to 8°C for up to 48 hours prior to • Abbott mSample Preparation • Abbott mSample Preparation
processing. System (4 × 24 Preps) System (4 × 24 Preps)
Human plasma (ACD-A and EDTA) specimens may be used with the (List No. 04J70-24) (List No. 04J70-24)
Abbott RealTime HIV-1 assay. Follow the manufacturer’s instructions for • Reaction Vessels • 5 mL Reaction Vessels
processing plasma collection tubes. • 20 µL to 1000 µL aerosol • Calibrated precision pipettes
To prepare EDTA and ACD-A plasma specimens, follow the barrier pipette tips for precision capable of delivering 20 to 1000
manufacturer’s instructions for processing plasma collection tubes. pipettes µL (Calibrated precision pipettes
After plasma preparation, plasma may be stored at 15 to 30°C for up • 11.6 to 16 mm Sample Tubes capable of delivering < 20 µL may
to 24 hours or at 2 to 8°C for up to 5 days. Plasma specimens may be be required if using UNG.)
stored at -20 +/- 10°C for up to 60 days. If longer storage is required, • Calibrated precision pipettes • Aerosol barrier pipette tips for
plasma specimens must be kept at –70°C or colder.33,34 Multiple capable of delivering 20 to 20 to 1000 μL pipettes (Aerosol
freeze-thaw cycles should be avoided. If frozen, thaw plasma specimens 1000 µL barrier pipette tips capable
at 15 to 30°C or at 2 to 8°C. Once thawed, if plasma specimens are not of delivering < 20 µL may be
being processed immediately, they can be stored at 2 to 8°C for up to required if using UNG.)
6 hours. • 200 µL and 1000 µL • 11.5 to 16 mm Sample Tubes
NOTE: Plasma specimens should not be frozen in non-gel blood disposable tips
collection tubes. • Abbott 96 Deep-Well Plate • 200 µL and 1000 µL disposable
• To prepare DBS, use finger prick or EDTA whole blood (not ACD (List No. 04J71-30) tips
whole blood). If EDTA whole blood needs to be shipped or stored • Vortex Mixer • Vortex Mixer
before spotting, the whole blood sample should be maintained under • Abbott Optical Adhesive • Abbott Optical Adhesive Covers
controlled temperature conditions (Refrigerated 2-8°C storage and Covers (List No. 04J71-75) (List No. 04J71-75)
shipment for no more than 48 hours. If 15-30°C temperature is used, • Abbott Adhesive Cover • Abbott Adhesive Cover
it should not exceed 30°C and 24 hours). Before spotting, mix the Applicators Applicators
blood using a pipette. Spot the blood onto the one-half-inch (12 • Abbott Splash-Free Support • Abbott Splash-Free Support Base
millimeter) circles on perforated Munktell paper card (or equivalent Base (List No. 09K31-01) (List No. 09K31-01)
such as Whatman 903 and Ahlstrom 226), ensuring that the entire • Reagent Troughs • Master Mix Tube (List No. 04J71-
circle is covered. It is recommended that at least 70 µL of blood 80)
(approximately 3 to 5 blood drops) be used for each circle to ensure • 1.5 mL Output Tubes • 200 mL Reagent Vessels
full coverage. • Centrifuge capable of 5000g • Abbott 96-Deep-Well Plate
• Air dry the card at ambient temperature. (List No. 04J71-30)
• Package each card in a sealable plastic bag with 2 to 3 desiccant • Uracil-N-glycosylase (UNG) • Uracil-N-glycosylase (UNG)
packs. The cards can be stored under ambient conditions for up to (List No. 06L87-02) (Optional) (List No. 06L87-02) (Optional)
8 weeks. Under conditions of high humidity (85%), the cards can be • Abbott 96-Well Optical Reaction
stored under ambient temperature for up to 2 weeks. Alternatively, Plate (List No. 04J71-70)
cards can be stored at 2 to 8°C or –10°C or colder for up to 12 • 1.4 mL Micro Vial 15 mm Caps
weeks. (List No. 3N20-01) optional
Specimen Transport • Centrifuge capable of 2000g
Ship specimens according to the recommended storage temperature • Abbott RealTime HIV-1 m2000
and time listed in the Specimen Collection and Storage section ROW System Combined
above. For domestic and international shipments, specimens should be Application CD-ROM (List No.
packaged and labeled in compliance with applicable state, federal, and 1L68-014 or higher)
international regulations covering the transport of clinical, diagnostic, or Additional materials required if
biological specimens. using DBS Sample Type:
NOTE: If EDTA whole blood needs to be shipped or stored before • 15.8 mm well diameter heat block
spotting, the whole blood sample should be maintained (to fit 15 mm diameter Master
under controlled temperature conditions (Refrigerated 2-8°C Mix Tubes)
storage and shipment for no more than 48 hours. If 15-30°C • Abbott mSample Preparation
temperature is used, it should not exceed 30°C and 24 hours). System DBS Buffer Kit (List No.
ABBOTT REALTIME HIV-1 ASSAY PROCEDURE 09N02-001)
This Abbott RealTime HIV-1 package insert contains 3 assay protocols: • m2000 System 13mm DBS PoST
• Plasma samples prepared for amplification using the Abbott m1000 Set (List No. 09N03-001)
System or the manual sample preparation method follow ASSAY For Abbott m1000 System For Abbott m2000rt Instrument
PROTOCOL I. Reagent Preparation Area Amplification Area
• Plasma Samples prepared for amplification using the Abbott • StrataCooler® 96 Benchtop • Abbott m2000rt instrument
m2000sp instrument follow ASSAY PROTOCOL II. Cooler or Eppendorf PCR
The Abbott RealTime HIV-1 assay provides 4 sample volume options Cooler
(0.2 mL, 0.5 mL, 0.6 mL, and 1.0 mL). (See assay Protocol II step 6 and • Abbott 96-Well Optical • Abbott RealTime HIV-1 m2000
RESULTS FOR PLASMA SPECIMENS section). Reaction Plate ROW System Combined
• DBS Samples prepared for amplification using the Abbott m2000sp (List No. 04J71-70) Application CD-ROM (List No.
instrument follow ASSAY PROTOCOL III. • Calibrated precision pipettes 1L68-014 or higher)
Materials Provided capable of delivering 20 to
1000 µL
• Abbott RealTime HIV-1 Amplification Reagent Kit
• 20 µL to 1000 µL aerosol • Abbott m2000rt Optical
(List No. 02G31-010)
barrier pipette tips for precision Calibration Kit (List No. 04J71-93)
Materials Required But Not Provided pipettes
• Abbott RealTime HIV-1 Calibrator Kit (List No. 2G31-70) • Single-use RNase/DNase-free
• Abbott RealTime HIV-1 Control Kit (List No. 2G31-80) tube or container
• Abbott mSample Preparation System DBS Buffer Kit • Vortex Mixer
(List No. 09N02-001) (if using DBS Sample Type)
For manual sample preparation method refer to the Materials and
Equipment Required Section of the Manual Sample Preparation for
Abbott RealTime RNA Assays Procedure (List No. 06L73).
5
Other Materials NOTE: Use 1 set of sample preparation reagent bottles, 1 vial of IC,
• Biological safety cabinet approved for working with infectious and 1 Abbott RealTime HIV-1 Amplification Reagent Pack to
materials support up to 24 reactions. Use a second set of reagents to
• Sealable plastic bags support 25 to 48 reactions. A maximum of 48 reactions can be
performed per run using an Abbott m1000 instrument.
• RNase-free water (Eppendorf or equivalent)†
• 1.7 mL molecular biology grade microcentrifuge tubes (Dot Sample Preparation Area
Scientific, Inc. or equivalent)† For sample preparation using the Abbott m1000 System, follow steps
• Cotton Tip Applicators (Puritan or equivalent)† 3 through 10. For the manual sample preparation method refer to the
†Note: These 3 items are used in the procedure for Monitoring the Extraction Protocol Section of the Manual Sample Preparation for Abbott
Laboratory for the Presence of Contamination. Refer to the RealTime RNA Assays Procedure (List No. 06L73).
QUALITY CONTROL PROCEDURES section of this package 3. Gently invert the Abbott mSample Preparation bottles to ensure a
insert. homogeneous solution. If crystals are observed in any of the reagent
bottles upon opening, allow the reagent to equilibrate at room
Procedural Precautions temperature until the crystals disappear. Do not use the reagents
• Read the instructions in this package insert carefully before until the crystals have dissolved.
processing samples.
4. Vortex each IC 3 times for 2 to 3 seconds before use.
• Amplification reagents and internal control (IC) may be used up to
2 times, as described in this package insert. The Abbott RealTime 5. Use a calibrated precision PIPETTE DEDICATED FOR INTERNAL
HIV-1 Calibrators, Negative Control, Low Positive Control, and High CONTROL USE ONLY to add 500 µL of IC to each bottle of mLysis
Positive Control vials are intended for single-use only and should be Buffer. Mix by gently inverting the container 5 to 10 times to minimize
discarded after use. foaming.
• Use aerosol barrier pipette tips or disposable pipettes only one time 6. A total of 48 samples can be processed in each run. A negative
when pipetting specimens, IC, or amplification reagents. To prevent control, a low positive control, and a high positive control are
contamination to the pipette barrel while pipetting, care should be included in each run, therefore allowing a maximum of 45 specimens
taken to avoid touching the pipette barrel to the inside of the sample to be processed per run.
tube or container. The use of extended aerosol barrier pipette tips is • The Abbott RealTime HIV-1 assay minimum sample volume and
recommended. associated rack requirements on the Abbott m1000 System are:
• Monitoring procedures for the presence of amplification product can
Abbott RealTime HIV-1
be found in the QUALITY CONTROL PROCEDURES section in this
Minimum Sample Volume
package insert.
Assay Application
• To reduce the risk of nucleic acid contamination, clean and
disinfect spills of specimens by including the use of a tuberculocidal Rack Tube Diametera 0.2 mL 0.5 mL 1.0 mL
disinfectant such as 1.0% sodium hypochlorite or other suitable 13 mm 11.6 mm - 14.0 mm 0.7 mL 1.0 mL 1.5 mL
disinfectant. 16 mm 15.0 mm - 16.0 mm 1.0 mL 1.3 mL 1.8 mL
• The Abbott RealTime HIV-1 Calibrators and Controls must be a Refers to sample tube outer diameter
prepared in conjunction with the specimens to be tested. The use
of the Abbott RealTime HIV-1 Controls and Calibrators is integral to • If frozen, thaw specimens at 15 to 30°C or at 2 to 8°C. Once
the performance of the Abbott RealTime HIV-1 assay. Refer to the thawed, specimens can be stored at 2 to 8°C for up to 6 hours if
QUALITY CONTROL PROCEDURES section of this package insert not processed immediately.
for details. NOTE: For every stored specimen, the following actions must
• IMPORTANT: Amplification reagents that will be used a second be done in the order described: vortex the specimen first
time must be stored at – 25 to – 15°C within 50 minutes of the and follow with centrifugation. If these actions are not
initiation of the master mix addition protocol. performed in this order, then invalid results may occur.
ASSAY PROTOCOL I: ABBOTT m1000 SYSTEM OR • Vortex each specimen 3 times for 2 to 3 seconds.
THE MANUAL SAMPLE PREPARATION METHOD AND • Centrifuge specimens at 2000g for 5 minutes before loading onto
the Abbott m1000 worktable. Aliquot each specimen into clean
ABBOTT m2000rt INSTRUMENT tubes or vials if necessary. Refer to the Abbott m1000 Operating
For a detailed description of how to perform an Abbott m1000 System Manual for tube sizes. Avoid touching the inside of the cap when
and Abbott m2000rt instrument protocol, refer to the Abbott m1000 opening tubes.
Operating Manual, Operation section and the Abbott m2000rt Operations 7. Place the calibrators (if applicable), low and high positive controls,
Manual, Operating Instructions section. the negative control, and the patient specimens into the Abbott
Laboratory personnel must be trained to operate the Abbott m1000 m1000 sample rack. Follow directions for performing a user‑defined
System and the Abbott m2000rt instrument. The operator must have a protocol, as described in the Abbott m1000 Operating Manual,
thorough knowledge of the software applications and must follow good Operation section.
laboratory practices.
8. Place the Reaction Vessels into the Abbott m1000 1 mL subsystem
For plasma samples prepared for amplification using the Abbott m1000 carrier.
System or the manual sample preparation method and using the optional
UNG procedure, refer to Appendix 2. 9. Load the Abbott mSample Preparation System reagents and the
1.5 mL Output Tubes on the Abbott m1000 System worktable as
1. Thaw assay controls and IC at 15 to 30°C or at 2 to 8°C. Thaw
described in the Abbott m1000 Operating Manual, Operation section.
calibrators at 15 to 30°C or at 2 to 8°C only if performing a
calibration run; see QUALITY CONTROL PROCEDURES section of 10. Initiate the Abbott m1000 protocol as described in the Abbott m1000
this package insert. Operating Manual, Operation section. From the Protocol screen,
select the appropriate application file corresponding to the sample
• Once thawed, assay controls, IC, and calibrators can be stored at
volume being tested.
2 to 8°C for up to 24 hours before use.
• The assembly of the amplification master mix and sample
• Vortex each assay calibrator and each control 3 times for 2 to 3
eluates into the Abbott 96-Well Optical Reaction Plate (step
seconds before use. Ensure that the contents of each vial are at
17) must be initiated within 1 hour after completion of Sample
the bottom after vortexing by tapping the vials on the bench to
Preparation.
bring liquid to the bottom of the vial.
2. Thaw amplification reagents at 15 to 30°C or at 2 to 8°C and store at Amplification Area
2 to 8°C until required for the amplification master mix procedure. 11. Switch on and initialize the Abbott m2000rt instrument.
• Once thawed, the amplification reagents can be stored at 2 to 8°C NOTE: The Abbott m2000rt instrument requires 15 minutes to
for up to 24 hours if not used immediately. warm up.

6
12. Create the Abbott m2000rt test order. Refer to the Operating 4. Clean the Splash-Free Support Base before next use, according to
Instructions section of the Abbott m2000rt Operations Manual. the Abbott m2000rt Operations Manual.
From the Protocol screen, select the appropriate application file 5. For manual sample preparation method users, refer to the Clean Up
corresponding to the sample volume being tested. Section of the Manual Sample Preparation for Abbott RealTime RNA
• Enter calibrator (needed if a calibration curve has not been stored Assays Procedure (List No. 06L73).
on the Abbott m2000rt) and control lot specific values in the test
order for accurate calibration and control evaluation. Lot-specific
ASSAY PROTOCOL II: PLASMA SAMPLES USING
values are specified in each Abbott RealTime HIV-1 Calibrator and THE ABBOTT m2000sp AND THE ABBOTT
Control Kit Card. m2000rt INSTRUMENTS
Reagent Preparation Area For a detailed description of how to perform an Abbott m2000sp
instrument and Abbott m2000rt instrument protocol, refer to the
All reagent preparation must take place in the dedicated Reagent
Abbott m2000sp and Abbott m2000rt Operations Manuals, Operating
Preparation Area. Refer to the Handling Precautions section of this
Instructions sections. The m2000sp protocol run requires Abbott
package insert before preparing reagents.
m2000sp Software Version 6.0 or higher. Please follow Abbott m2000sp
NOTE: Change gloves before handling the amplification reagents.
Operations Manual (List 9K20) version 6 or higher.
13. Prepare the amplification master mix.
Laboratory personnel must be trained to operate the Abbott m2000sp
• Each Amplification Reagent Pack supports up to 24 reactions. and Abbott m2000rt instruments. The operator must have a thorough
• Prior to opening the amplification reagents, ensure that the knowledge of the applications run on the instruments and must follow
contents of the vials are at the bottom by tapping the vials in an good laboratory practices.
upright position on the bench 5 to 10 times to bring the liquid to For plasma Samples prepared for amplification using the Abbott
the bottom of the vials. m2000sp instrument and using the optional UNG procedure, refer to
• Prepare the master mix by using a PIPETTE DEDICATED FOR Appendix 2.
REAGENT USE ONLY to add 271 µL of the HIV-1 Activation 1. Thaw assay controls and IC at 15 to 30°C or at 2 to 8°C. Thaw
Reagent (Reagent 1) and 949 µL of the HIV-1 Oligonucleotide calibrators at 15 to 30°C or at 2 to 8°C only if performing a
Reagent (Reagent 2) together in the Thermostable rTth DNA calibration run; see QUALITY CONTROL PROCEDURES section of
Polymerase Enzyme bottle (Reagent 3). this package insert.
• If performing 25 to 48 reactions, prepare a second amplification • Once thawed, assay controls, IC, and calibrators can be stored at
master mix with a second Amplification Reagent Pack. 2 to 8°C for up to 24 hours before use.
• The Abbott m2000rt protocol (step 20) must be initiated within • Vortex each assay calibrator and each control 3 times for 2 to 3
40 minutes of the addition of Activation Reagent into the first seconds before use. Ensure that the contents of each vial are at
rTth Enzyme Reagent bottle (step 13). the bottom after vortexing by tapping the vials on the bench to
14. Pipette the contents of the master mix from the enzyme bottle(s) bring liquid to the bottom of the vial.
into a single-use RNase/DNase-free tube and vortex to mix. 2. Select new and/or partial amplification reagent packs to be used in
15. Place an Abbott 96-Well Optical Reaction Plate in a StrataCooler the run. Refer to the Abbott m2000sp Operations Manual
96 or Eppendorf PCR Cooler stored as indicated in the instruction (List No. 9K20 version 6 or higher), Operating Instructions section,
manual. Using a DEDICATED PIPETTE, dispense 50 µL aliquots of for instructions pertaining to amplification reagent pack inventory
the amplification master mix into the Abbott 96-Well Optical Reaction management. Amplification reagent packs must have the same
Plate. A calibrated repeat pipettor may be used. Visually verify that lot number.
50 µL has been dispensed into each well. Thaw new amplification reagents at 15 to 30°C or at 2 to 8°C and
16. Transfer the Abbott 96-Well Optical Reaction Plate on the store at 2 to 8°C until required for the amplification master mix
StrataCooler 96 or Eppendorf PCR Cooler to the Sample procedure. Once thawed, the new amplification reagents can be
Preparation Area. stored at 2 to 8°C for up to 24 hours if not used immediately.
NOTE: Partial amplification reagent packs being used a second
Sample Preparation Area time should NOT be stored at 2 to 8°C before use. They
17. In the Sample Preparation Area, transfer 50 µL of sample eluate to should be kept at – 25 to – 15°C until needed for master
the Abbott 96-Well Optical Reaction Plate on the StrataCooler 96 or mix addition. Once removed from the freezer, cumulative
Eppendorf PCR Cooler. Use a separate pipette tip for each sample room temperature exposure should not exceed 25 minutes,
eluate transfer. During the transfer of each sample, mix the reaction including instances where packs are removed from storage,
by pipetting up and down 3 to 5 times. Visually verify that 100 µL has but not used. If 25 minutes is exceeded, discard the partial
been dispensed into each well. amplification reagent packs.
18. Seal the Abbott 96-Well Optical Reaction Plate according to the The following table shows the number of sample preparation reagents
instructions in the Abbott m2000rt Operations Manual. and internal control vials needed based on the number of reactions.
19. Remove the Abbott 96-Well Optical Reaction Plate from the
Sample Preparation Reagents and Internal Control Requirements
StrataCooler 96 or Eppendorf PCR Cooler and place in the Abbott
Splash-Free Support Base. Centrifuge the Abbott 96-Well Optical 1 to 24 25 to 48 49 to 72 73 to 96
Reaction Plate in the Abbott Splash-Free Support Base at 5,000g for Reagent Reactions Reactions Reactions Reactions
5 minutes. Transfer to the Amplification Area. mMicroparticles 1 bottle 2 bottles 2 bottles 2 bottles
NOTE: Do not transfer the StrataCooler 96 or Eppendorf PCR
Cooler to the Amplification Area. mLysis 1 bottle 2 bottles 3 bottles 4 bottles
Amplification Area mWash 1 1 bottle 2 bottles 3 bottles 4 bottles
20. Place the Abbott 96-Well Optical Reaction Plate in the Abbott mWash 2 1 bottle 2 bottles 3 bottles 4 bottles
m2000rt instrument. From the Protocol screen, select the appropriate
mElution Buffer 1 bottle 2 bottles 3 bottles 4 bottles
application file corresponding to the sample volume being tested.
Initiate the Abbott RealTime HIV-1 protocol, as described in the Internal Control a 1 new 1 new 2 new 2 new
Abbott m2000rt Operations Manual, Operating Instructions section. vial or vial or vials or vials or
1 partial 2 partial 3 partial 4 partial
POST PROCESSING PROCEDURES FOR PROTOCOL I vial vials vials vials
1. Clean the StrataCooler 96 or Eppendorf PCR Cooler as described in
the instruction manual and return to the Reagent Preparation Area. a A combination of new and partial vials of Internal Control may be
2. Remove the 1.5 mL Output Tubes from the worktable and dispose of used.
according to the Abbott m1000 Operating Manual. 3. Gently invert the Abbott mSample Preparation bottles to ensure a
3. Place the Abbott 96-Well Optical Reaction Plate in a sealable plastic homogeneous solution. If crystals are observed in any of the reagent
bag and dispose of according to the Abbott m2000rt Operations bottles upon opening, allow the reagent to equilibrate at room
Manual along with the gloves used to handle the plate. temperature until the crystals disappear. Do not use the reagents
until the crystals have dissolved.

7
4. Vortex each IC 3 times for 2 to 3 seconds before use.
Amplification Reagent Pack Requirements a
5. Use a calibrated precision PIPETTE DEDICATED FOR INTERNAL
CONTROL USE ONLY to add 500 µL of IC to each bottle of mLysis 1 to 24 25 to 48 49 to 72 73 to 96
Buffer. Mix by gently inverting the container 5 to 10 times to minimize Reactions Reactions Reactions Reactions
foaming. Partial vials of IC can be recapped and stored at – 25 to 1 if new; 2 if new; 3 if new; 4 new
– 15°C for a second use. up to 4 with up to 4 with up to 4 with or
6. A total of 96 samples can be processed in each run, with the partial packs partial packs partial packs partial packs
exception of the 1.0 mL Assay Application. A negative control, a a Refer to the Abbott m2000sp Operations Manual (List No. 9K20 version 6 or
low positive control, and a high positive control are included in each higher) for instructions on inventory management to determine the maximum
run, therefore allowing a maximum of 93 specimens to be processed number of reactions that can be tested with the partial packs selected.
per run. For the 1.0 mL Assay Application, a total of 48 samples can • Partial amplification reagent packs can only be used on the
be processed in each run, allowing a maximum of 45 specimens to same Abbott m2000sp instrument used for the reagent pack’s
be processed per run. initial preparation. Using an amplification reagent pack for a
• The Abbott RealTime HIV-1 assay minimum sample volume and second time on a different instrument will result in an error,
associated rack requirements on the Abbott m2000sp are: which may delay the run.
• Partial and new amplification reagent packs may be
Abbott RealTime HIV-1
used together.
Minimum Sample Volume
Assay Application IMPORTANT: Partial amplification reagent packs should be stored at
– 25 to – 15°C until immediately before the second use. Confirm that
Rack Tube Diametera 0.2 mL 0.5 mL 0.6 mL 1.0 mL
master mix is thawed before placing partial pack(s) on the Abbott
13 mm 11.5 - 14.0 mm 0.4 - 0.8 mL 0.7 - 1.2 mL 0.8 - 1.3 mL 1.2 - 1.7 mL m2000sp worktable. Once removed from – 25 to – 15°C storage,
16 mm 14.5 - 16.0 mm 0.4 - 1.0 mL 0.8 - 1.4 mL 0.9 - 1.5 mL 1.3 - 1.9 mL partial amplification reagent packs being used a second time must be
a Refers to sample tube outer diameter. Minimum sample volume varies with used within 25 minutes or discarded. This applies to cumulative room
tube geometry and size. Refer to the Abbott m2000sp Operations Manual and temperature exposure, including instances where packs are removed
QUICK REFERENCE GUIDE FOR SAMPLE TUBE SIZES AND VOLUMES for from storage, but not used.
recommended sample input volume. • Ensure that the contents of new amplification reagent packs
are at the bottom of the vials prior to opening the amplification
• If frozen, thaw specimens at 15 to 30°C or at 2 to 8°C. Once
reagents by tapping the vials in an upright position on the bench
thawed, specimens can be stored at 2 to 8°C for up to 6 hours if
5 to 10 times.
not processed immediately.
• Do not tap partial amplification reagent packs being used a
NOTE: For every stored specimen, the following actions must
second time. Tapping may result in loss of master mix volume in
be done in the order described: vortex the specimen first
the cap.
and follow with centrifugation. If these actions are not
performed in this order, then invalid results may occur. • Remove caps. If a new amplification reagent pack will be stored
for a second use, the vials will need to be recapped for storage.
• Vortex each specimen 3 times for 2 to 3 seconds.
If planning to reuse the original caps to recap the reagent vials,
• Centrifuge specimens at 2000g for 5 minutes before loading save the original caps. If planning to use fresh caps to recap the
onto the Abbott m2000sp worktable. Aliquot each specimen into reagent vials, original caps may be discarded.
clean tubes or vials if necessary. Refer to the Abbott m2000sp
• Partial amplification reagent packs are loaded to the left of new
Operations Manual for tube sizes. Avoid touching the inside of the
amplification reagent packs on the Abbott m2000sp worktable.
cap when opening tubes.
• Ensure that amplification reagent packs are firmly seated on
7. Place the low and high positive controls, the negative control, the
the instrument.
calibrators, if applicable, and the patient specimens into the Abbott
m2000sp sample rack. If used, bar codes on tube labels must face 12. Select the appropriate deep-well plate that matches the
right for scanning. corresponding sample preparation extraction. Initiate the Abbott
m2000sp Master Mix Addition protocol. Follow the instructions as
8. Place the 5 mL Reaction Vessels into the Abbott m2000sp 1 mL described in the Abbott m2000sp Operations Manual, Operating
subsystem carrier. Instructions section.
9. Load the Abbott mSample Preparation System reagents and the NOTE: The operator should not manually fill any empty/unfilled
Abbott 96 Deep-Well Plate on the Abbott m2000sp worktable as wells in the Abbott 96-Well Optical Reaction Plate.
described in the Abbott m2000sp Operations Manual, Operating
• After sample extraction is complete, the Abbott m2000sp
Instructions section.
automatically fills any empty wells in the Abbott 96-Well Optical
10. From the Protocol screen, select the appropriate application file Reaction Plate when there are greater than 48 samples processed
corresponding to the sample volume being tested. Initiate the sample within a run. Plate fill is not performed for runs containing 48
extraction protocol as described in the Abbott m2000sp Operations samples or fewer.
Manual, Operating Instruction section. • If prompted by the instrument, Reagent Carrier 2 should remain
• Enter calibrator (needed if a calibration curve has not been stored in place, minimally containing the reagent vessel for mElution
on the Abbott m2000rt) and control lot specific values in the Buffer (Reagent Carrier 2, location 6). If this reagent vessel has
Sample Extraction: Worktable Setup, Calibrator and Control been unloaded, place a new reagent vessel with the mElution
fields. Lot-specific values are specified in each Abbott RealTime Buffer label into Reagent Carrier 2, location 6. System fluid will
HIV-1 Calibrator and Control Kit Card. be added to the reagent vessel and used to fill empty wells. Once
• The Abbott m2000sp Master Mix Addition protocol (step 12) this process is complete, the system will continue with the master
must be initiated within 1 hour after completion of Sample mix addition.
Preparation. NOTE: System instructions for use of the automated plate-filling
NOTE: Change gloves before handling the amplification feature are found in the Abbott m2000sp Operations
reagents. Manual (List No. 9K20 version 6 or higher), section
11. Load the amplification reagents and the master mix tube (if needed) 5, Operating Instructions, Sample Extraction—Closed
on the Abbott m2000sp worktable after sample preparation is Mode.
completed. The following table shows the number of amplification • The Abbott m2000rt protocol (step 16) must be started within
reagent packs needed based on the number of reactions. 50 minutes of the initiation of the Master Mix Addition protocol
If only 1 amplification reagent pack is being used, no master mix (step 12).
tube is required. NOTE: If the run is aborted for any reason subsequent to step
12, a new 96-well PCR plate must be used if the Abbott
m2000sp Master Mix Addition Protocol (step 12) will be
repeated.

8
13. Switch on and initialize the Abbott m2000rt instrument in the 5. Manually shake or swirl the sample tubes and then place them in a
Amplification Area. heat block set at 55°C. Do not vortex the samples. Incubate for 30
NOTE: The Abbott m2000rt requires 15 minutes to warm-up. minutes (± 2 minutes) at 55°C.
NOTE: Remove gloves before returning to the sample preparation 6. Meanwhile, thaw assay controls and internal control (IC) at 15 to
area. 30°C or at 2 to 8°C. Thaw calibrators at 15 to 30°C or at 2 to 8°C
14. Seal the Abbott 96-Well Optical Reaction Plate after the Abbott only if performing a calibration run.
m2000sp instrument has completed addition of samples and master NOTE: Once thawed, assay controls, IC, and calibrators can be stored
mix according to the Abbott m2000sp Operations Manual, Operating at 2 to 8°C for up to 24 hours before use.
Instructions section. 7. Vortex each assay calibrator and each control 3 times for 2 to 3
15. Place the sealed optical reaction plate into the Abbott Splash-Free seconds before use. Ensure that the contents of each vial are at
Support Base for transfer to the Abbott m2000rt instrument. the bottom by tapping the vials on the bench to bring liquid to the
bottom of the vial. Ensure bubbles or foam is not generated; if
16. Place the Abbott 96-Well Optical Reaction Plate in the Abbott present, remove with a sterile pipette tip, using a new tip for each
m2000rt instrument. From the Protocol screen, select the appropriate vial.
application file corresponding to the sample volume being tested.
Initiate the Abbott RealTime HIV-1 protocol, as described in the 8. Thaw amplification reagents at 15 to 30°C or at 2 to 8°C and store at
Abbott m2000rt Operations Manual, Operating Instructions section. 2 to 8°C until required for the amplification master mix procedure.
NOTE: Test order transfer through the use of CD-ROM or network 9. Select new and/or partial amplification reagent packs to be used in
connection with export and import features of the m2000sp the run. Refer to the Abbott m2000sp Operations Manual
and m2000rt software is recommended. If creating the (List No. 9K20 version 6 or higher), Operating Instructions section,
Abbott m2000rt test order manually, enter sample IDs in the for instructions pertaining to amplification reagent pack inventory
corresponding PCR tray locations according to the “Wells management. Amplification reagent packs must have the same
for Selected Plate” grid, found on the detail screen of the lot number.
“PCR Plate Results” on the Abbott m2000sp. See Section Thaw new amplification reagents at 15 to 30°C or at 2 to 8°C and
5 of the Abbott m2000sp Operations Manual. store at 2 to 8°C until required for the amplification master mix
procedure. Once thawed, the new amplification reagents can be
17. If a prepared partial amplification reagent pack is to be used a
stored at 2 to 8°C for up to 24 hours if not used immediately.
second time, cap the 3 reagent vials with the saved caps or new
caps (List No. 3N20-01) and promptly store the reagents at – 25 to NOTE: Partial amplification reagent packs being used a second
– 15°C, protected from light, and in an upright position. Discard any time should NOT be stored at 2 to 8°C before use. They
amplification reagent packs that are exhausted or have been used should be kept at – 25 to – 15°C until needed for master
twice. mix addition. Once removed from the freezer, cumulative
IMPORTANT: Amplification reagents that will be used a second time room temperature exposure should not exceed 25 minutes,
must be stored at – 25 to – 15°C within 50 minutes of the initiation of including instances where packs are removed from storage,
the master mix addition protocol. but not used. If 25 minutes is exceeded, discard the partial
amplification reagent packs.
ASSAY PROTOCOL III: DBS SAMPLES USING The following table shows the number of sample preparation reagents
THE ABBOTT m2000sp AND THE ABBOTT and internal control vials needed based on the number of reactions.
m2000rt INSTRUMENTS
Sample Preparation Reagents and Internal Control Requirements
For a detailed description of how to perform an Abbott m2000sp
instrument and Abbott m2000rt instrument protocol, refer to the 1 to 24 25 to 48 49 to 72 73 to 96
Abbott m2000sp and Abbott m2000rt Operations Manuals, Operating Reagent Reactions Reactions Reactions Reactions
Instructions sections. The DBS protocol requires Abbott m2000sp mMicroparticles 1 bottle 2 bottles 2 bottles 2 bottles
Software Version 6.0 or higher. Please follow Abbott m2000sp
Operations Manual (List 9K20 version 6 or higher). mLysis 1 bottle 2 bottles 3 bottles 4 bottles
A total of 96 samples can be processed in each run. A negative mWash 1 1 bottle 2 bottles 3 bottles 4 bottles
control, a low positive control, and a high positive control are included
in each run, therefore allowing a maximum of 93 DBS specimens mWash 2 1 bottle 2 bottles 3 bottles 4 bottles
to be processed per run when calibrators are not included. Process mElution Buffer 1 bottle 2 bottles 3 bottles 4 bottles
Calibrators and Controls directly as liquid samples; step 1 through step
Internal Control a 1 new 1 new 2 new 2 new
5 are for DBS only. Do not process plasma specimens on any run where
vial or vial or vials or vials or
DBS protocol is used. For each DBS sample, a single Abbott Master
1 partial 2 partial 3 partial 4 partial
Mix Tube should be used for the entire sample processing procedure.
vial vials vials vials
Ensure DBS samples are labeled throughout processing. a A combination of new and partial vials of Internal Control may be used.
For DBS Samples prepared for amplification using the Abbott m2000sp
instrument and using the optional UNG procedure follow, refer to 10. Gently invert the Abbott mSample Preparation bottles to ensure a
Appendix 2. homogeneous solution. If crystals are observed in any of the reagent
1. Fill the Abbott Master Mix Tube with 1.3 mL of mDBS buffer from the bottles upon opening, allow the reagent to equilibrate at room
Abbott mSample Preparation system DBS Buffer Kit (List No. 09N02- temperature until the crystals disappear. Do not use the reagents
001). until the crystals have dissolved.
NOTE: Do not use mLysis Buffer or any other reagents for this 11. Vortex each IC 3 times for 2 to 3 seconds before use.
step. 12. Use a calibrated precision pipette DEDICATED FOR INTERNAL
2. Hold perforated DBS paper card above the Abbott Master Mix Tube. CONTROL USE ONLY to add 750 µL of IC to each bottle of mLysis
Buffer. Mix by gently inverting the container 5 to 10 times to minimize
3. Push the DBS circle out of the card using a clean pipette tip, one
foaming.
DBS circle per Master Mix Tube. Each DBS should be approximately
one-half-inch (12 millimeters) in diameter. USE A NEW PIPETTE TIP 13. Place the low and high positive controls, the negative control, and
FOR EACH DBS SAMPLE TO PREVENT CROSS CONTAMINATION. the calibrators, if applicable, into the Abbott m2000sp sample racks.
4. Ensure that the DBS circle is fully submerged in the mDBS Buffer 14. After the incubation is complete, manually shake or swirl the DBS
by tapping the tube or using the pipette tip to push the DBS into the sample tubes and then place them into the Abbott m2000sp sample
buffer. racks.
NOTE: If a pipette tip is used to push the DBS into the buffer, ensure NOTE: Ensure that the Abbott m2000sp sample racks have been
that the pipette tip does not cause DBS buffer volume loss due calibrated specifically for the Abbott RealTime HIV-1 DBS
to liquid containment in the tip and/or absorption of the buffer procedure.
by the tip filter.

9
15. Load the sample racks carefully to avoid splashing. If used, bar 23. Select the appropriate deep-well plate that matches the
codes on tube labels must face right for scanning. Ensure that each corresponding sample preparation extraction. Initiate the Abbott
tube is placed securely in the sample rack so that the bottom of the m2000sp Master Mix Addition protocol. Follow the instructions as
tube reaches the inside bottom of the rack. described in the Abbott m2000sp Operations Manual, Operating
16. Load filled sample racks onto the Abbott m2000sp in consecutive Instructions section.
sample rack positions, with the first rack farthest to the right on the NOTE: The operator should not manually fill any empty/unfilled
worktable, and any additional rack progressively to the left of the wells in the Abbott 96-Well Optical Reaction Plate.
first rack. • After sample extraction is complete, the Abbott m2000sp
17. Place the 5 mL Reaction Vessels into the Abbott m2000sp 1 mL automatically fills any empty wells in the Abbott 96-Well Optical
subsystem carrier. Reaction Plate with mElution buffer when there are greater than
48 samples processed within a run. Plate fill is not performed for
18. Load the Abbott mSample Preparation System reagents and the
runs containing 48 samples or fewer.
Abbott 96 Deep-Well Plate on the Abbott m2000sp worktable as
described in the Abbott m2000sp Operations Manual, Operating • If prompted by the instrument, Reagent Carrier 2 should remain
Instructions section. in place, minimally containing the reagent vessel for mElution
Buffer (Reagent Carrier 2, location 6). If this reagent vessel has
19. From the Protocol screen, select the HIV-1 DBS viral load application been unloaded, place a new reagent vessel with the mElution
file. Initiate the sample extraction protocol as described in the Abbott Buffer label into Reagent Carrier 2, location 6. System fluid will
m2000sp Operations Manual, Operating Instruction section. be added to the reagent vessel and used to fill empty wells. Once
20. Enter calibrator (needed if a calibration curve has not been stored this process is complete, the system will continue with the master
on the Abbott m2000rt) and control lot specific values in the mix addition.
Sample Extraction: Worktable Setup, Calibrator and Control fields. NOTE: System instructions for use of the automated plate-filling
Lot-specific values are specified in each Abbott RealTime HIV-1 feature are found in the Abbott m2000sp Operations
Calibrator and Control Kit Card. Manual (List No. 9K20 version 6 or higher), section 5,
21. The Abbott m2000sp Master Mix Addition protocol (step 23) must be Operating Instructions, Sample Extraction-Closed Mode.
initiated within 1 hour after completion of Sample Preparation. • The Abbott m2000rt protocol (step 27) must be started within 50
NOTE: Change gloves before handling the amplification reagents. minutes of the initiation of the Master Mix Addition protocol
22. Load the amplification reagents and the master mix tube (if needed) (step 23).
on the Abbott m2000sp worktable after sample preparation is NOTE: If the run is aborted for any reason subsequent to step 23,
completed. The following table shows the number of amplification a new Abbott 96-Well Optical Reaction Plate must be used
reagent packs needed based on the number of reactions. if the Abbott m2000sp Master Mix Addition Protocol (step
If only 1 amplification reagent pack is being used, no master mix 23) will be repeated.
tube is required. 24. Switch on and initialize the Abbott m2000rt instrument in the
Amplification Area.
Amplification Reagent Pack Requirements a NOTE: The Abbott m2000rt requires 15 minutes to warm-up.
1 to 24 25 to 48 49 to 72 73 to 96 NOTE: Remove gloves before returning to the sample preparation
Reactions Reactions Reactions Reactions area.
1 if new; 2 if new; 3 if new; 4 new 25. Seal the Abbott 96-Well Optical Reaction Plate after the Abbott
up to 4 with up to 4 with up to 4 with or m2000sp instrument has completed addition of samples and master
partial packs partial packs partial packs partial packs mix according to the Abbott m2000sp Operations Manual, Operating
a Instructions section.
Refer to the Abbott m2000sp Operations Manual (List No. 9K20 version 6 or
higher) for instructions on inventory management to determine the maximum 26. Place the sealed optical reaction plate into the Abbott Splash-Free
number of reactions that can be tested with the partial packs selected. Support Base for transfer to the Abbott m2000rt instrument.
• Partial amplification reagent packs can only be used on the same 27. Place the Abbott 96-Well Optical Reaction Plate in the Abbott
Abbott m2000sp instrument used for the reagent pack’s initial m2000rt instrument. From the Protocol screen, select the HIV-1 DBS
preparation. Using an amplification reagent pack for a second viral load application file. Initiate the protocol as described in the
time on a different instrument will result in an error, which may Abbott m2000rt Operations Manual, Operating Instructions section.
delay the run. NOTE: Test order transfer through the use of CD-ROM or network
• Partial and new amplification reagent packs may be connection with export and import features of the m2000sp
used together. and m2000rt software is recommended. If creating the
IMPORTANT: Partial amplification reagent packs should be stored Abbott m2000rt test order manually, enter sample IDs in the
at – 25 to – 15°C until immediately before the second use. corresponding PCR tray locations according to the “Wells for
Confirm that master mix is thawed before placing partial Selected Plate” grid, found on the detail screen of the “PCR
pack(s) on the Abbott m2000sp worktable. Once removed Plate Results” on the Abbott m2000sp. See Section 5 of the
from – 25 to – 15°C storage, partial amplification reagent packs Abbott m2000sp Operations Manual.
being used a second time must be used within 25 minutes 28. If a prepared partial amplification reagent pack is to be used a
or discarded. This applies to cumulative room temperature second time, cap the 3 reagent vials with the saved caps or new
exposure, including instances where packs are removed from caps (List No. 3N20-01) and promptly store the reagents at – 25 to
storage, but not used. – 15°C, protected from light, and in an upright position. Discard any
• Ensure that the contents of new amplification reagent packs amplification reagent packs that are exhausted or have been used
are at the bottom of the vials prior to opening the amplification twice.
reagents by tapping the vials in an upright position on the bench IMPORTANT: Amplification reagents that will be used a second time
5 to 10 times. must be stored at – 25 to – 15°C within 50 minutes of the
• Do not tap partial amplification reagent packs being used a initiation of the master mix addition protocol.
second time. Tapping may result in loss of master mix volume in
the cap.
POST PROCESSING PROCEDURES FOR PROTOCOL II
• Remove caps. If a new amplification reagent pack will be stored
AND III
for a second use, the vials will need to be recapped for storage. 1. Remove the Abbott 96 Deep-Well Plate from the worktable and
If planning to reuse the original caps to recap the reagent vials, dispose of according to the Abbott m2000sp Operations Manual.
save the original caps. If planning to use fresh caps to recap the 2. Place the Abbott 96-Well Optical Reaction Plate in a sealable
reagent vials, original caps may be discarded. plastic bag and dispose according to the Abbott m2000rt Operations
• Partial amplification reagent packs are loaded to the left of new Manual along with the gloves used to handle the plate.
amplification reagent packs on the Abbott m2000sp worktable. 3. Clean the Abbott Splash-Free Support Base before next use,
• Ensure that amplification reagent packs are firmly seated on the according to the Abbott m2000rt Operations Manual.
instrument.

10
QUALITY CONTROL PROCEDURES The presence of HIV-1 must not be detected in the negative control.
HIV‑1 detected in the negative control is indicative of contamination
Abbott m2000rt Optical Calibration by other samples or by amplified product introduced during sample
Refer to the Calibration Procedures section in the Abbott m2000rt preparation or during preparation of the Abbott 96-Well Optical Reaction
Operations Manual for a detailed description of how to perform an Plate. To avoid contamination, clean the Abbott m1000 System or Abbott
Abbott m2000rt Optical Calibration. m2000sp instrument and the Abbott m2000rt instrument and repeat
Optical calibration of the Abbott m2000rt instrument is required for the sample processing for controls and specimens following the Procedural
accurate measurement and discrimination of dye fluorescence during Precautions. If negative controls are persistently reactive, contact your
the Abbott RealTime HIV-1 assay. Abbott representative.
The following Abbott m2000rt Optical Calibration Plates are used to Monitoring the Laboratory for the Presence of Contamination
calibrate the Abbott m2000rt instrument for the Abbott RealTime HIV-1
It is recommended that this test be done at least once a month to
assay:
monitor laboratory surfaces and equipment for contamination by
• FAM™ Plate (Carboxyfluorescein) amplification product. It is very important to test all areas that may have
• ROX™ Plate (Carboxy-X-rhodamine) been exposed to processed specimens, controls, and calibrators, and/
• VIC® Plate (Proprietary dye) or amplification product. This includes routinely handled objects such
Assay Calibration as pipettes, the Abbott m2000sp and Abbott m2000rt function keys,
laboratory bench surfaces, microcentrifuges, and centrifuge adaptors.
For a detailed description of how to perform an assay calibration refer to
the Abbott m2000sp and Abbott m2000rt Operations Manuals, Operating 1. Add 0.8 mL RNase-free water to a 1.7 mL molecular biology grade
Instructions sections. microcentrifuge tube.
A calibration curve is required to quantitate the HIV-1 RNA concentration 2. Saturate the cotton tip of an applicator (Puritan or equivalent) in the
of specimens and controls. Two assay calibrators are run in replicates RNase-free water from the microcentrifuge tube.
of 3 to generate a calibration curve (logarithm of HIV-1 concentration 3. Using the saturated cotton tip of the applicator, wipe the area to be
versus the threshold cycle [CT] at which a reactive level of fluorescent monitored using a sweeping motion. Place the applicator into the
signal is detected). The calibration curve slope and intercept are microcentrifuge tube.
calculated and stored on the instrument. The concentration of HIV-1 RNA 4. Swirl the cotton tip in RNase-free water 10 times, and then press the
in a sample is calculated from the stored calibration curve. Results are applicator along the inside of the tube so that the liquid drains back
automatically reported on the Abbott m2000rt workstation. into the solution at the bottom of the microcentrifuge tube. Discard
Follow the procedure for sample extraction, master mix addition, the applicator.
amplification and detection protocols as stated in the Abbott m2000sp
Operations Manual, and the Abbott m2000rt Operations Manual. 5. Pipette 0.5 mL of mWash 1 buffer to a clean tube using the pipette
dedicated for Internal Control use.
Once an Abbott RealTime HIV-1 calibration is accepted and stored, it
may be used for 6 months. During this time, all subsequent samples 6. Add 20 µL of the mWash 1 buffer to each microcentrifuge tube.
may be tested without further calibration unless: 7. Cap the microcentrifuge tube.
• An Abbott RealTime HIV-1 Amplification Reagent Kit with a new lot 8. Test this sample according to the assay procedure section of this
number is used. package insert.
• An Abbott mSample Preparation System (4 × 24 Preps) with a new • Transfer liquid from the microcentrifuge tube to a 5 mL Reaction
lot number is used. Vessel.
• An Abbott RealTime HIV-1 application file for a different sample • Bring the volume to 1.5 mL with RNase-free water.
volume is used.
9. The presence of contamination is indicated by the detection of HIV-1
• A new Abbott RealTime HIV-1 application specification file is nucleic acid in the swab samples.
installed.
10. If HIV-1 nucleic acid is detected on equipment, follow the cleaning
• Pure Dye optical re-calibration of the Abbott RealTime HIV-1 assay-
and decontaminating guidelines given in that equipment’s operations
specific dyes (FAM, VIC, or ROX) is performed per the Calibration
manual. If HIV-1 nucleic acid is detected on surfaces, clean the
Procedures section of the Abbott m2000rt Operations Manual.
contaminated areas with 1.0% (v/v) sodium hypochlorite solution,
Detection of Inhibition followed by 70% ethanol or water.
An IC threshold cycle [CT] assay validity parameter is established during NOTE: Chlorine solutions may pit equipment and metal. Use
a calibration run. sufficient amounts or repeated applications of 70% ethanol
A defined, consistent quantity of IC is introduced into each specimen, or water until chlorine residue is no longer visible.
calibrator, and control at the beginning of sample preparation and 11. Repeat testing of the contaminated area by following steps 1
measured on the Abbott m2000rt instrument to demonstrate proper through 10.
specimen processing and assay validity. The IC is comprised of an RNA
sequence unrelated to the HIV-1 target sequence. RESULTS FOR PLASMA SPECIMENS
The median amplification cycle at which the IC target sequence Calculation
fluorescent signal is detected in calibration samples establishes an IC CT The concentration of viral HIV-1 RNA in a sample or control is calculated
validity range to be met by all subsequent processed specimens. from the stored calibration curve. The Abbott m2000rt instrument
An error control flag is displayed when a specimen or control fails to automatically reports the results on the Abbott m2000rt workstation.
meet this specification. Refer to the Abbott m2000rt Operations Manual Assay results can be reported in copies/mL, log [copies/mL],
for an explanation of the corrective actions for the error control flag. International Units (IU)/mL, or log [IU/mL]; (1 IU = 0.58 copies,
Specimens whose IC CT value exceeds the established range must be 1 copy = 1.74 IU).
retested starting with sample preparation.
Negative and Positive Controls
A negative control, a low-positive control, and a high-positive control are
included in each test order to evaluate run validity.
The lot-specific values for the low-positive control and high-positive
control are specified on each Abbott RealTime HIV-1 Control Kit Card
and must be entered into the assay test order when a run is performed.
An error control flag is displayed when a control result is out of range.
Refer to the Abbott m2000rt Operations Manual for an explanation of
the corrective actions for the error control flag. If negative or positive
controls are out of range, all of the specimens and controls from that
run must be reprocessed, beginning with sample preparation.

11
Interpretation of Results SPECIFIC PERFORMANCE CHARACTERISTICS FOR
Sample Volume Result Interpretation PLASMA SPECIMENS
1.0 mL Not Detected Target not detected The performance characteristics were determined using the Abbott
RealTime HIV-1 assay with Abbott m2000sp sample preparation and 1.0
< 1.60 Log [Copies/mL]a Detected
mL sample volume, unless otherwise specified.
1.60 to 7.00 Log [Copies/mL]
> 7.00 Log [Copies/mL] > ULQd
Limit of Detection (LOD)
The limit of detection is defined as the HIV-1 RNA concentration
0.6 mL Not Detected Target not detected
detected with a probability of 95% or greater.
< 1.60 Log [Copies/mL]a Detected
1.60 to 7.00 Log [Copies/mL] Limit of Detection, 1.0 mL Sample Volume
> 7.00 Log [Copies/mL] > ULQd The LOD of the Abbott RealTime HIV-1 assay is 40 copies/mL with the
1.0 mL sample volume procedure.
0.5 mL Not Detected Target not detected
The LOD was determined by testing dilutions of a viral standard from
< 1.88 Log [Copies/mL]b Detected
the Virology Quality Assurance (VQA) Laboratory of the AIDS Clinical
1.88 to 7.00 Log [Copies/mL] Trial Group. Dilutions were made in HIV-1 negative human plasma.
> 7.00 Log [Copies/mL] > ULQ Testing was performed with 3 lots of amplification reagents on 3 Abbott
0.2 mL Not Detected Target not detected m2000 Systems. The results, representative of the analytical sensitivity
< 2.18 Log [Copies/mL]c Detected performance of the Abbott RealTime HIV-1 assay, are summarized in
2.18 to 7.00 Log [Copies/mL] Table 1.
> 7.00 Log [Copies/mL] > ULQ Table 1.
a 40 Copies/mL
b
Conc. Number Number Percent
75 Copies/mL (Copies/mL) Tested Detected Detected
c 150 Copies/mL
d 100 57 57 100
ULQ = upper limit of quantitation
75 57 57 100
RESULTS FOR DBS SPECIMENS
60 57 57 100
The reported sample concentration result from the m2000rt DBS
protocol run represents the HIV-1 viral concentration in the plasma of 50 57 57 100
the whole blood specimen from which the DBS specimen is obtained. 40 57 57 100
The Abbott m2000rt instrument automatically reports the results on the 30 57 55 96
Abbott m2000rt workstation. Assay results can be reported in copies/mL,
20 57 50 88
log [copies/mL], International Units (IU)/mL, or log [IU/mL]; (1 IU = 0.58
copies, 1 copy= 1.74 IU). 10 56a 38 68
5 57 30 53
Interpretation of Results a One replicate generated an invalid replicate error message and was deleted from
Result Interpretation the data analysis.
Not Detected Target not detected Probit analysis of the data determined that the concentration of HIV-1
<2.92 Log [Copies/mL]a Detected RNA detected with 95% probability was 25 copies/mL (95% CI 20 to 33).
2.92 to 7.00 Log [Copies/mL]
Limit of Detection, 0.6 mL Sample Volume
>7.00 Log [Copies/mL] > ULQb The LOD of the Abbott RealTime HIV-1 assay is 40 copies/mL with the
a 839 Copies/mL
b ULQ = upper limit of quantitation
0.6 mL sample volume procedure.
The LOD for the 0.6 mL sample volume procedure was determined
The concentration values for the controls and calibrators provided in as described for the 1.0 mL sample volume procedure. The results,
their kit cards represent the HIV-1 target concentrations in these plasma representative of the analytical sensitivity performance of the Abbott
equivalent samples. When an assay run is performed with the DBS RealTime HIV-1 assay, are summarized in Table 2.
protocol, the concentration values reported for controls and calibrators
will reflect DBS equivalents. This scenario has no impact on results for Table 2.
DBS specimens. Conc. Number Number Percent
LIMITATIONS OF THE PROCEDURE (Copies/mL) Tested Detected Detected
• FOR IN VITRO DIAGNOSTIC USE 100 57 57 100
• Optimal performance of this test requires appropriate specimen 75 57 56 98
collection, storage, and transport to the test site (refer to the 60 57 57 100
SPECIMEN COLLECTION, STORAGE, AND TRANSPORT TO THE 50 57 54 95
TEST SITE section of this package insert). 40 57 54 95
• Whole blood specimens for human plasma (collected in ACD-A or 30 57 55 96
EDTA tubes) and DBS (collected in EDTA tubes) may be used with 20 57 44 77
the Abbott RealTime HIV-1 assay. The use of other anticoagulants 10 57 27 47
has not been validated with the Abbott RealTime HIV-1 assay. 5 57 13 23
• Use of the Abbott RealTime HIV-1 assay is limited to personnel who Probit analysis of the data determined that the concentration of HIV-1
have been trained in the procedures of a molecular diagnostic assay RNA detected with 95% probability was 39 copies/mL (95% CI 33 to 49).
and the Abbott m1000 and Abbott m2000 systems.
Limit of Detection, 0.5 mL Sample Volume
• The instruments and assay procedures reduce the risk of
The LOD of the Abbott RealTime HIV-1 assay is 75 copies/mL with the
contamination by amplification product. However, nucleic acid
0.5 mL sample volume procedure.
contamination from the calibrators, positive controls, or specimens
must be controlled by good laboratory practices and careful The LOD for the 0.5 mL sample volume procedure was determined
adherence to the procedures specified in this package insert. as described for the 1.0 mL sample volume procedure. The results,
representative of the analytical sensitivity performance of the Abbott
• As with any diagnostic test, results from the Abbott RealTime HIV-1
RealTime HIV-1 assay, are summarized in Table 3.
assay should be interpreted in conjunction with other clinical and
laboratory findings. A specimen with a result of “Not Detected”
cannot be presumed to be negative for HIV-1 RNA.

12
 Table 3. Figure 1.
Conc. Number Number Percent
(Copies/mL) Tested Detected Detected
100 57 57 100
75 57 57 100
60 57 54 95
50 56a 52 93
40 57 47 82
30 57 46 81
20 57 42 74
10 57 26 46
5 57 21 37
a One replicate generated an invalid replicate error message and was deleted from
the data analysis.
Probit analysis of the data determined that the concentration of HIV-1
RNA detected with 95% probability was 65 copies/mL (95% CI 51 to 88).
Limit of Detection, 0.2 mL Sample Volume
The LOD of the Abbott RealTime HIV-1 assay is 150 copies/mL with the
0.2 mL sample volume procedure.
The LOD for the 0.2 mL sample volume procedure was determined The Abbott RealTime HIV-1 assay was shown to be linear across the
as described for the 1.0 mL sample volume procedure. The results, range tested (n = 99, r = 0.999, slope = 0.93, and intercept = 0.26).
representative of the analytical sensitivity performance of the Abbott
RealTime HIV-1 assay, are summarized in Table 4. Precision
The precision of the Abbott RealTime HIV-1 assay was evaluated for
Table 4. the 1.0 mL sample volume procedure using the Abbott m1000 and
Conc. Number Number Percent Abbott m2000sp sample preparation systems and the manual sample
(Copies/mL) Tested Detected Detected preparation method. The Abbott RealTime HIV-1 assay is designed to
250 57 57 100 achieve an inter-assay standard deviation (SD) of less than or equal
to 0.25 log copies of HIV-1 RNA per mL for samples containing HIV-1
200 57 56 98 concentrations from 500 to 5 million copies/mL. A 7‑member HIV-1 RNA
150 57 56 98 panel was prepared by diluting an HIV-1 viral stock (panel members
100 57 54 95 1 through 3) and armored HIV-1 RNA (panel members 4 through 7) in
75 57 47 82 negative human plasma. For the precision studies with the Abbott m1000
and the Abbott m2000sp, the panel members were tested in replicates
60 57 38 67 of 5 in a total of 15 runs on 3 instrument systems, with 3 lots of
50 57 39 68 amplification reagents. For the precision study using the manual sample
40 54a 30 56 preparation method, panel members were tested in replicates of 2 for
30 52a 19 37 the first run on each instrument and replicates of 3 for each subsequent
a run for a total of 15 runs on 3 Abbott m2000rt instruments with 3 lots of
Eight replicates were invalid due to an instrument error and were deleted from
the data analysis. amplification reagents. Precision analysis was performed following the
NCCLS EP10-A2 guideline.36 Within‑run, between‑run, and inter-assay
Probit analysis of the data determined that the concentration of HIV-1 (within-run and between-run) standard deviations were determined. The
RNA detected with 95% probability was 119 copies/mL (95% CI 102 to results, representative of the precision of the Abbott RealTime HIV-1
150). assay, are summarized in Tables 5, 6, and 7.
Linear Range Table 5.
The upper limit of quantitation (ULQ) for the Abbott RealTime HIV‑1
Precision with the Abbott m1000 System
assay is 10 million copies/mL, and the lower limit of quantitation
Within‑Run Between‑Run
is equivalent to the LOD (40 copies/mL for the 1.0 mL and 0.6 mL Panel Conc. Mean Conc. Mean SD SD Inter-Assay
sample volume procedure, 75 copies/mL for the 0.5 mL sample Member n (copies/mL) (log copies/mL) Component Component SDa
volume procedure, and 150 copies/mL for the 0.2 mL sample volume 1 75 57 1.75 0.21 0.00 0.21
procedure). 2 75 573 2.76 0.08 0.00 0.08
A 9-member panel prepared by diluting armored HIV-1 RNA from 7.44 log 3 75 5,000 3.70 0.05 0.02 0.06
copies/mL to 1.16 log copies/mL in HIV-1 negative human plasma was
4 73b,c 35,751 4.55 0.03 0.01 0.04
tested. Linearity analysis was performed following the NCCLS EP6-A
guideline.35 The results, representative of the Abbott RealTime HIV-1 5 75 315,065 5.50 0.07 0.03 0.07
assay linearity, are shown in Figure 1. 6 74b 2,947,538 6.47 0.05 0.04 0.07
7 75 5,347,285 6.73 0.04 0.05 0.07
a Inter-assay contains within-run and between-run components.
b Two replicates were inhibited and were deleted from the data analysis.
c HIV-1 RNA was not detected in 1 replicate.

13
 The specificity of the assay was further evaluated by testing 70
Table 6.
specimens that had been either obtained from individuals diagnosed or
Precision with the Abbott m2000 System screened for an autoimmune disorder or serologically characterized as
Within‑Run Between‑Run positive for the following markers: systemic lupus erythematosus (SLE),
Panel Conc. Mean Conc. Mean SD SD Inter-Assay
anti‑nuclear antibodies (ANA), rheumatoid factor (RF), HBsAg, anti-
Member n (copies/mL) (log copies/mL) Component Component SDa
HTLV-I/II, anti-HCV, and anti‑HIV‑2. HIV-1 RNA was not detected in any
1 74b 72 1.86 0.18 0.07 0.19 of the specimens tested. The results demonstrated that the presence of
2 75 652 2.81 0.08 0.00 0.08 an autoimmune disorder or serologic markers for autoimmune disease
3 75 5,417 3.73 0.04 0.02 0.05 or viral pathogens other than HIV-1 did not affect the Abbott RealTime
4 75 39,458 4.60 0.04 0.03 0.05 HIV-1 assay.
5 74c 358,587 5.55 0.03 0.03 0.04 Cross-Reactivity
6 75 3,102,654 6.49 0.03 0.02 0.04 The following viruses and microorganisms were evaluated for potential
7 75 5,953,879 6.77 0.04 0.04 0.05 cross-reactivity in the Abbott RealTime HIV-1 assay. Purified nucleic acid
a Inter-assay contains within-run and between-run components. or viral lysate from each microorganism or virus was added to HIV‑1 RNA
b negative samples and samples that contained 10,000 copies/mL HIV-1
HIV-1 RNA was not detected in 1 replicate.
c
RNA.
One replicate was inhibited and was deleted from the data analysis. Human Immunodeficiency virus 2 Vaccinia virus
Human T-lymphotropic virus 1 BK human polyomavirus
Table 7. Hepatitis C virus Human papilloma virus 16
Precision with Manual Sample Preparation Method Hepatitis B virus Human papilloma virus 18
Within‑Run Between‑Run Epstein-Barr virus Neisseria gonorrhoeae
Panel Conc. Mean Conc. Mean SD SD Inter-Assay
Herpes simplex virus 1 Chlamydia trachomatis
Member n (copies/mL) (log copies/mL) Component Component SDa
Herpes simplex virus 2 Candida albicans
1 40b 46 1.66 0.21 0.07 0.22
Cytomegalovirus Staphylococcus aureus
2 41c 471 2.67 0.11 0.09 0.14 Human herpesvirus 6B Staphylococcus epidermidis
3 42 4,474 3.65 0.05 0.10 0.11 Human herpesvirus 8 Mycobacterium gordonae
4 42 34,503 4.54 0.02 0.06 0.07 Varicella-zoster virus Mycobacterium smegmatis
5 42 362,283 5.56 0.04 0.08 0.09 No interference in the performance of the Abbott RealTime HIV-1 assay
6 42 3,597,099 6.56 0.03 0.04 0.05 was observed in the presence of the potential cross-reactants for all
7 42 6,552,825 6.82 0.05 0.05 0.07 positive and negative samples tested.
a Inter-assay contains within-run and between-run components. Detection of HIV-1 Subtypes and Groups
b HIV-1 RNA was not detected in 2 replicates. The performance of the Abbott RealTime HIV-1 assay with HIV-1
c One replicate was inhibited and deleted from the data analysis. subtypes/groups was evaluated by analysis of purified RNA transcripts
from Group M (subtypes A, B, C, D, CRF01-AE, F, CRF02-AG, G, and
Potentially Interfering Substances H), Group O, and Group N, and by testing 10 clinical specimens of each
The susceptibility of the Abbott RealTime HIV-1 assay to interference Group M subtype (A, B, C, D, CRF01-AE, F, CRF02-AG, and G), and 10
by elevated levels of endogenous substances and by drugs commonly specimens from Group O.
prescribed to HIV-1 infected individuals was evaluated. HIV-1 negative RNA transcripts of Group M (subtypes A, B, C, D, CRF01-AE, F, CRF02-
samples and samples containing 10,000 copies/mL of HIV-1 RNA were AG, G, and H), Group O, and Group N with concentrations targeted to
tested. approximately 6.0 log copies/mL, 4.7 log copies/mL, 3.0 log copies/
No interference in the performance of the Abbott RealTime HIV-1 assay mL, and 1.7 log copies/mL were tested. Three replicates were tested at
was observed in the presence of the following substances for all positive each concentration for each transcript. The results, representative of the
and negative samples tested: dilution linearity for the 11 subtypes/groups tested, are shown in
• Hemoglobin 500 mg/dL Figure 2.
• Triglycerides 3000 mg/dL Figure 2.
• Bilirubin 20 mg/dL
• Protein 9 g/dL
Drugs at concentrations in excess of the peak plasma or serum levels
were tested in 5 pools. No interference in the performance of the Abbott
RealTime HIV-1 assay was observed in the presence of the following
drug pools for all positive and negative samples tested:
Drug Pool Drugs Tested
1 Zidovudine, Saquinavir, Ritonavir, Clarithromycin, Interferon
2a, Interferon 2b
2 Abacavir sulfate, Amprenavir, Peginterferon 2a,
Peginterferon 2b, Ribavirin
3 Tenofovir disoproxil fumarate, Lamivudine, Indinavir sulfate,
Ganciclovir, Valganciclovir hydrochloride, Acyclovir
4 Stavudine, Efavirenz, Lopinavir, Enfuvirtide, Ciprofloxacin
5 Zalcitabine, Nevirapine, Nelfinavir, Azithromycin,
Valacyclovir
Specificity
The target specificity of the Abbott RealTime HIV-1 assay is greater than
or equal to 99.5% after resolution.
The specificity of the Abbott RealTime HIV-1 assay was evaluated by
testing 187 HIV-1 seronegative plasma specimens. The specimens were
tested on 3 Abbott m2000 instrument systems with 3 lots of amplification
reagents. HIV-1 RNA was not detected, resulting in 100% (187/187)
specificity (95% CI 98.05 to 100.00) in this representative study.

14
The results showed that all subtypes and groups tested were detected,
Figure 4.
and dilution linearity was demonstrated for all groups and subtypes
tested (correlation coefficients ranged from 0.997 to 1.000).
A total of 90 clinical specimens, 10 of each Group M subtype (A, B,
C, D, CRF01-AE, F, CRF02‑AG, G) and Group O, were tested with
the Abbott RealTime HIV-1 assay and by 2 other HIV-1 quantitative
assays referred to as Comparator 1 and Comparator 2. The results are
summarized in Table 8.
Table 8.
Group/ RealTime Comparator 1 Comparator 2
Subtypes n Detected Detecteda Detecteda
M/Subtype A 10 10 10 (1) 10 (1)
M/Subtype B 10 10 10 (0) 10 (0)
M/Subtype C 10 10 10 (0) 10 (0)
M/Subtype D 10 10 10 (0) 10 (0)
M/Subtype AE 10 10 10 (0) 10 (0)
M/Subtype F 10 10 10 (0) 10 (0)
M/Subtype AG 10 10 10 (3) 10 (1)
SPECIFIC PERFORMANCE CHARACTERISTICS FOR DBS
M/Subtype G 10 10 10 (2) 10 (1)
SPECIMENS
Group O 10 10 0 (NA) 7 (7)
a The numbers in parentheses are the number of specimens that had lower
Limit of Detection
quantitation values by more than 1.00 log copies/mL when compared to Abbott The LOD of the Abbott RealTime HIV-1 assay is 839 copies/mL with the
RealTime HIV-1 assay. DBS sample type.
• The Abbott RealTime HIV-1 assay detected all subtypes and groups The limit of detection was determined by analysis of an HIV-1 viral
tested. dilution series from the VQA (Virology Quality Assurance laboratory)
standard. Twenty-eight samples at each concentration level were tested
• Comparator 1 detected all Group M subtypes tested and did not across 4 runs using 4 lots of amplification reagents. The detection
detect the 10 Group O samples. rate for each dilution panel member was summarized across the four
• Comparator 2 detected all Group M subtypes tested and 7 out of 10 lots of reagents. The results, representative of the analytical sensitivity
Group O samples. performance of the Abbott RealTime HIV-1 assay, are summarized in
• There were no samples that had Abbott RealTime assay quantitation Table 9.
values lower than Comparator 1 or Comparator 2 values by more
than 1.00 log copies/mL. Table 9.
• There were 6 Group M samples that had lower quantitation values Conc. Number Number Percent
with Comparator 1 by more than 1.00 log/copies/mL when compared (Copies/mL) Tested Detected Detected
to Abbott RealTime HIV-1 assay. 3000 27a 27 100
• There were 3 Group M samples and 7 Group O samples that had 1000 28 27 96
lower quantitation values with Comparator 2 by more than 1.00 log 500 28 24 86
copies/mL when compared to Abbott RealTime HIV-1 assay.
250 28 10 36
Correlation 125 28 4 14
Method comparison analysis was performed following NCCLS EP9-A2.37
a One replicate was invalid and was excluded from the analysis.
Specimens from 141 HIV-1 infected patients were tested with the Abbott
RealTime HIV-1 assay and a comparator assay. The correlation plot is Probit analysis of the data determined that the concentration of HIV-1
shown in Figure 3. RNA detected with 95% probability was 839 copies/mL (95% CI 624 to
1387 copies/mL).
Figure 3.
An additional limit of detection study was performed by testing
another HIV-1 viral dilution series. A minimum of 36 samples at
each concentration level were tested across 15 runs using 1 lot of
amplification reagents. The detection rate for each dilution panel
member was summarized across the runs. The results, representative
of the analytical sensitivity performance of the Abbott RealTime HIV-1
assay, are summarized in Table 10.
Table 10.
Conc. Number Number Percent
(Copies/mL) Tested Detected Detected (%)
5012 36 36 100
2512 36 36 100
1000 59a,b 57 97
501 60a 49 82
251 36 13 36
a 24 additional replicates were run for these panel members.
b One replicate was invalid and was excluded from the analysis.

Probit analysis of the data determined that the concentration of HIV-1

RNA detected with 95% probability was 828 copies/mL (95% CI 671 to
1192 copies/mL).
Specimens from 79 HIV-1 infected patients (a subset of the 141 tested)
were tested with the Abbott LCx HIV RNA Quantitative assay. The
correlation plot is shown in Figure 4.

15
Linear Range Table 11.
The upper limit of quantitation (ULQ) for the Abbott RealTime HIV-1
Abbott RealTime HIV-1 Precision for Dried Blood Spot
assay is 10 million copies/mL and the lower limit of quantitation is
Conc. Mean Within‑Run Between‑Run
equivalent to the LOD (839 copies/mL) for the DBS claim. Panel Conc. Mean (log copies/ SD SD Inter-Assaya
A dilution series of HIV-1 Armored RNA covering the range from 500 Member n (copies/mL) mL) Component Component SD
copies/mL to 10,000,000 copies/mL in HIV-1 sero-negative blood was 1 54b 417 2.62 0.29 0.00 0.29
tested. The results, representative of the Abbott RealTime HIV-1 assay 2 70b 692 2.84 0.26 0.00 0.26
linearity, are shown in Figure 5.
3 74c 4531 3.66 0.12 0.09 0.16
Figure 5. 4 73d 9034 3.96 0.11 0.07 0.13
5 75 108643 5.04 0.05 0.04 0.06
6 75 8130801 6.91 0.05 0.04 0.06
a Inter-assay contains within-run and between-run components.
b Concentration means of Panel Members 1 and 2 are below LOD and the
precision estimates reflect only results that are quantitated and, therefore, are for
information only.
c One replicate was invalid and was excluded from the data analysis.
d Two replicates were invalid and were excluded from the data analysis.

Precision was evaluated in an additional study by testing HIV-1 panel

members targeted to cover the range from 501 copies/mL to 10,000,000
copies/mL. One lot of amplification reagents was run on three pairs
of m2000 instrument systems, once a day for five days. Within‑run,
between‑run, and inter-assay (within-run and between-run) standard
deviations (SD) were determined. The results, representative of the
precision of the Abbott RealTime HIV-1 assay, are summarized in
Table 12.
Table 12.
Within‑Run Between‑Run
The Abbott RealTime HIV-1 assay was shown to be linear across the Panel Conc. Mean Conc. Mean SD SD Inter-Assaya
range tested (n = 92, r = 0.995, slope = 1.08, and intercept = -0.32). Member n (copies/mL) (log copies/mL) Component Component SD
1 49b 444 2.65 0.23 0.12 0.26
An additional dilution series was tested using an HIV-1 viral stock
covering the range from 501 copies/mL to 10,000,000 copies/mL in 2 57c 984 2.99 0.28 0.06 0.29
HIV-1 sero-negative blood. The results, representative of the Abbott 3 60 10977 4.04 0.11 0.10 0.15
RealTime HIV-1 assay linearity, are shown in Figure 6. 4 60 125458 5.10 0.08 0.05 0.09
5 58d 19786971 7.30 0.06 0.06 0.09
Figure 6. a Inter-Assay contains within-run and between-run components.
b Concentration mean of Panel Member 1 is below LOD and the precision estimates
Abbott RealTime HIV DBS (Log copies/mL)

reflect only results that are quantitated and therefore are for information only.
cOne replicate was invalid and was excluded from the data analysis and two replicates
were not detected.
d Two replicates were invalid and were excluded from the data analysis.

Specificity
The target specificity of the Abbott RealTime HIV-1 assay is greater than
or equal to 99.5% after resolution.
Specificity was determined by testing 120 HIV-1 sero-negative
specimens, 60 specimens with each of two lots of amplification
reagents. All 120 HIV-1 sero-negative specimens gave results of “Not
Detected” for a specificity of 100% (120/120).
Correlation
HIV-1 RNA quantitation was compared between the Abbott RealTime HIV-
1 assay using dried blood spots and the CE-marked comparator assay,
Target Conc. (Log copies/mL) Abbott RealTime HIV-1 RNA quantitative assay using human plasma. A
total of 313 specimens collected from South Africa, Ivory Coast, and
The Abbott RealTime HIV-1 assay was shown to be linear across the range tested Uganda were included in the analysis. These HIV-1 infected patients
(n = 58, r = 0.994, slope = 1.09, and intercept = -0.40). were tested at Abbott Molecular (N=247) and at one external site in
South Africa (N=66). For each HIV-1 infected patient dried blood spots
Precision prepared from venous blood and capillary blood (finger prick) were
Precision was evaluated by testing HIV-1 panel members targeted to tested. The results from specimens that fell within the common assay
cover the range from 500 Copies/mL to 5,000,000 Copies/mL. Three dynamic range were analyzed by the least squares linear regression
lots of amplification reagents were run on the three pairs of m2000 method (DBS finger prick versus plasma N=150, DBS venous versus
instrument systems (each lot of reagent assigned to its own instrument plasma N=150, and DBS finger prick versus DBS venous N=146). The
pair), once a day for five days. Within‑run, between‑run, and inter-assay correlation coefficient for HIV-1 viral load in plasma versus DBS finger
(within-run and between-run) standard deviations (SD) were determined. prick was 0.887, the slope was 0.84 (95% CI 0.77 to 0.91), and the
The results, representative of the precision of the Abbott RealTime HIV-1 intercept was 0.58 log copies/mL (95% CI 0.26 to 0.90) (Figure 7). The
assay, are summarized in Table 11. correlation coefficient for HIV-1 viral load in plasma versus DBS venous
blood was 0.902, the slope was 0.83 (95% CI 0.76 to 0.89), and the
intercept was 0.66 log copies/mL (95% CI 0.37 to 0.95) (Figure 8). The
correlation coefficient for HIV-1 viral load in DBS finger prick versus DBS
venous blood was 0.947, the slope was 1.00 (95% CI 0.94 to 1.05), and
the intercept was –0.03 log copies/mL (95% CI –0.28 to 0.21) (Figure
9). Additionally, Bland-Altman plots for these same comparisons are
presented in Figure 10, Figure 11, and Figure 12.

16
Figure 7. DBS Finger Prick Versus Plasma Figure 10. DBS Finger Prick Versus Plasma

y=0.84x + 0.58 n=150

r=0.887 Mean of Bias -0.13
n=150

Figure 11. DBS Venous Versus Plasma

Figure 8. DBS Venous Versus Plasma
n=150
y=0.83x + 0.66 Mean of Bias -0.10
r=0.902
n=150

(Venous + Plasma) /2 (Log copies/mL)

Figure 12. DBS Finger Prick Versus DBS Venous

Figure 9. DBS Finger Prick Versus DBS Venous
n=146
y=1.00x - 0.03 Mean of Bias -0.05
r=0.947
n=146

An additional study was performed to compare HIV-1 RNA quantitation

between the Abbott RealTime HIV-1 assay using dried blood spots
and the CE-marked comparator assay, Abbott RealTime HIV-1 RNA
quantitative assay using human plasma. A total of 244 specimens were
included in the analysis. These HIV-1 infected patients were tested at
Abbott Molecular. One DBS test result from each patient was used to
compare to the result from matched plasma. Results from specimens
that fell within the common assay dynamic range were analyzed by the
least squares linear regression method (DBS versus plasma N=119).
The correlation coefficient for HIV-1 viral load in plasma versus DBS
was 0.929, the slope was 0.76 (95% CI 0.71 to 0.82), and the intercept
was 1.20 log copies/mL (95% CI 0.97 to 1.43) (Figure 13). Additionally, a
Bland-Altman plot is presented in Figure 14.

17
 10. Ho DD, Neumann AU, Perelson AS, et al. Rapid turnover of plasma
Figure 13. DBS vs. Plasma
virions and CD4 lymphocytes in HIV-1 infection. Nature 1995;373:123-6.
y=0.76x + 1.20 11. Wei X, Ghosh SK, Taylor ME, et al. Viral dynamics in human
r=0.929 immunodeficiency virus type 1 infection. Nature 1995;373:117‑22.
n=119 12. Mellors JW, Rinaldo CR JR, Gupta P, et al. Prognosis in HIV-1
DBS conc. (Log copies/mL) infection predicted by the quantity of virus in plasma. Science
1996;272:1167-70.
13. Mellors JW, Munoz A, Giorgi JV, et al. Plasma viral load and CD4+
lymphocytes as prognostic markers of HIV-1 infection. Ann Intern
Med 1997;126(12):946-54.
14. Chene G, Sterne JA, May M, et al. Prognostic importance of initial
response in HIV-1 infected patients starting potent antiretroviral
therapy: analysis of prospective studies. Lancet 2003;362:679-86.
15. Egger M, May M, Chene G, et al. Prognosis of HIV-1 infected drug
patients starting highly active antiretroviral therapy: a collaborative
analysis of prospective studies. Lancet 2002;360:119-29.
16. Wood E, Hogg RS, Yip B, et al. Higher baseline levels of plasma
human immunodeficiency virus type 1 RNA are associated with
Plasma conc. (Log copies/mL) increased mortality after initiation of triple-drug antiretroviral therapy.
J Infect Dis 2003;188:1421-5.
Figure 14. DBS vs. Plasma 17. US Department of Health and Human Services. 2004 guidelines
for the use of antiretroviral agents in HIV-1 infected adults and
adolescents. Available at: http://AIDSinfo.nih.gov/guidelines.
n=119
Mean of Bias=0.24 18. Yeni PG, Hammer SM, Hirsch MS, et al. Treatment for Adult HIV
Bias (DBS - Plasma) (Log copies/mL)

Infection. 2004 Recommendations of the International AIDS Society-

USA Panel. JAMA 2004;292:251-65.
19. Perelson AS, Essunger P, Cao Y, et al. Decay characteristics of
HIV-1 infected compartments during combination therapy. Nature
1997;387(6629):188-91.
20. Interim Technical Update. Technical and Operational Considerations
for Implementing HIV Viral Load Testing July 2014. WHO.
21. Mulder J, McKinney N, Christopher C, et al. Rapid and simple PCR
assay for quantitation of human immunodeficiency virus type 1 RNA
in plasma: application to acute retroviral infection. J Clin Microbiol
1994;32:292-300.
22. Dewar RL, Highbarger HC, Sarmiento MD, et al. Application
of branched DNA signal amplification to monitor human
immunodeficiency virus type 1 burden in human plasma. J Inf
(DBS + Plasma)/2 (Log copies/mL) Diseases 1994;170:1172‑9.
23. Van Gemen B, Kievits T, Schukkink R, et al. Quantification of HIV-1
RNA in plasma using NASBA™ during HIV-1 primary infection. J Virol
Methods 1993;43:177-87.
BIBLIOGRAPHY 24. Yen-Lieberman B, Brambilla D, Jackson B, et al. Evaluation
1. Barre-Sinoussi F, Chermann JC, Rey F, et al. Isolation of a of a quality assurance program for quantitation of human
T-lymphotropic retrovirus from a patient at risk for acquired immune immunodeficiency virus type 1 RNA in plasma by the AIDS clinical
deficiency syndrome (AIDS). Science 1983;220:868-71. trials group virology laboratories. J Clin Microbiol 1996;34:2695-701.
2. Popovic M, Sarngadharan MG, Read E, et al. Detection, isolation 25. Holmes H, Davis C, Heath A, et al. An international collaborative
and continuous production of cytopathic retroviruses (HTLV-I) from study to establish the 1st international standard for HIV-1 RNA for use
patients with AIDS and pre-AIDS. Science 1984;224:497-500. in nucleic acid-based techniques. J Virol Methods 2001;92:141-50.
3. Gallo RC, Salahuddin SZ, Popovic M, et al. Frequent detection and 26. Davis C, Heath A, Best S, et al. Calibration of HIV-1 working reagents
isolation of cytopathic retroviruses (HTLV-I) from patients with AIDS for nucleic acid amplification techniques against the 1st international
and at risk for AIDS. Science 1984;224:500-3. standard for HIV-1 RNA. J Virol Meth 2003;107:37-44.
4. Curran JW, Jaffe HW, Hardy AM, et al. Epidemiology of HIV infection 27. Myers TW, Gelfand DH. Reverse Transcription and DNA
and AIDS in the United States. Science 1988;239:610‑16. Amplification by a Thermus thermophilus DNA Polymerase. Biochem
1991;30:7661-6.
5. Daar ES, Moudgil T, Meyer RD, Ho DD. Transient high levels of
viremia in patients with primary human immunodeficiency virus type 28. Myers G, Korber B, Wain-Hobson S, et al. Human retroviruses and
1 infection. New Engl J Med 1991;324:961-4. AIDS 1994: A compilation and analysis of nucleic acid and amino
acid sequences. Los Alamos National Laboratory, Theoretical Biology
6. Clark SJ, Saag MS, Decker WD. High titers of cytopathic virus in
and Biophysics (T10). Los Alamos, New Mexico:1994.
plasma of patients with symptomatic primary HIV-1 infection. New
Engl J Med 1991;324:954-60. 29. US Department of Health and Human Services. Biosafety in
Microbiological and Biomedical Laboratories. 5th ed. Washington,
7. Albert J, Abrahamsson B, Nagy K, et al. Rapid development
DC: US Government Printing Office; December 2009.
of isolate-specific neutralizing antibodies after primary HIV-1
[Also available online. Type> www.cdc.gov, search>BMBL5>look up
infection and consequent emergence of virus variants which resist
sections III and IV.]
neutralization by autologous sera. AIDS 1990;4:107‑12.
8. Horsburgh CR Jr, Ou CY, Jason J, et al. Duration of human
immunodeficiency virus infection before detection of antibody.
Lancet 1989;334:637-40.
9. Pantaleo G, Graziosi C, Fauci AS. New concepts in the
immunopathogenesis of human immunodeficiency virus (HIV)
infection. New Engl J Med 1993;328:327-35.

18
30. US Department of Labor, Occupational Safety and Health The Abbott RealTime HIV-1 Amplification Reagent Kit is imported into the
Administration. 29 CFR Part 1910.1030. Bloodborne Pathogens. European Union by Abbott Diagnostics GmbH, located at Max-Planck-
31. Clinical and Laboratory Standards Institute. Protection of Laboratory Ring 2, 65205 Wiesbaden, Germany.
Workers from Occupationally Acquired Infections: Approved
Guideline—Third Edition. CLSI Document M29-A3. Wayne, PA:
Clinical and Laboratory Standards Institute; 2005.
32. World Health Organization. Laboratory Biosafety Manual. 3rd ed. 1434
Geneva, Switzerland: World Health Organization; 2004.
33. Ginocchio C, Wang X, Kaplan M, et al. Effects of specimen
collection, processing, and storage conditions on stability of human
immunodeficiency virus type 1 RNA levels in plasma. J Clin Microbiol
1997;35(11):2886‑93. © 2016, 2020 Abbott. All Rights Reserved.
34. Sebire K, McGavin K, Land S, et al. Stability of human www.abbottmolecular.com
immunodeficiency virus RNA in blood specimens as measured by a June 2020
commercial PCR-based assay. J Clin Microbiol 1998;36(2):493-8. 51-608282/R9
35. National Committee for Clinical Laboratory Standards. Evaluation of
the Linearity of Quantitative Measurement Procedures: A Statistical
Approach; Approved Guideline - NCCLS document EP6-A, NCCLS:
Wayne, PA, 2002.
36. National Committee for Clinical Laboratory Standards. Preliminary
Evaluation of Quantitative Clinical Laboratory Methods; Approved
Guideline – Second Edition. NCCLS Document EP10-A2. NCCLS:
Wayne, PA, 2002.
37. National Committee for Clinical Laboratory Standards. Method
Comparison and Bias Estimation Using Patient Samples; Approved
Guideline-Second Edition NCCLS document EP9-A2, NCCLS Wayne,
PA, 2002.
TECHNICAL ASSISTANCE
For technical assistance, call Abbott Molecular Technical Services at
1-800-553-7042 (within the US) or +49-6122-580 (outside the US), or
visit the Abbott Molecular website at www.molecular.abbott/portal.
THE PURCHASE OF THIS PRODUCT ALLOWS THE PURCHASER TO
USE IT FOR AMPLIFICATION OF NUCLEIC ACID SEQUENCES AND
FOR DETECTION OF NUCLEIC ACID SEQUENCES FOR HUMAN IN
VITRO DIAGNOSTICS. NO GENERAL PATENT OR OTHER LICENSE
OF ANY KIND OTHER THAN THIS SPECIFIC RIGHT OF USE FROM
PURCHASE IS GRANTED HEREBY. THIS PROVISION DOES NOT
PROHIBIT THE RESALE OF THIS PRODUCT.
Armored RNA® is a patented technology jointly developed by Ambion,
Inc. and Cenetron Diagnostics, LLC. US patents #5,677,124, #5,919,625,
#5,939,262 and patents pending.

Armored RNA is a registered trademark of Ambion.

ProClin is a registered trademark of Rohm and Haas.
StrataCooler is a registered trademark of Stratagene.
FAM and ROX are trademarks of Life Technologies Corporation or its
subsidiaries in the US and/or certain other countries.
VIC is a registered trademark of Life Technologies Corporation or its
subsidiaries in the US and/or certain other countries.
Abbott m, m1000, m2000, m2000rt, and m2000sp are trademarks of
Abbott Laboratories.
LCx is a registered trademark of Abbott Laboratories.
Abbott Molecular Inc. is the legal manufacturer of the:
Abbott RealTime HIV-1 Amplification Reagent Kit (List No. 02G31-010)
Abbott RealTime HIV-1 Control Kit (List No. 2G31-80)
Abbott RealTime HIV-1 Calibrator Kit (List No. 2G31-70).

19
APPENDIX 1. OVERVIEW OF THE ABBOTT REALTIME HIV-1 AMPLIFICATION REAGENT EXTENDED USE FEATURE
The amplification reagent extended use feature allows for the use of an amplification reagent pack and internal control (IC) a total of 2 times.
Amplification reagent packs that have not yet been used to prepare master mix are referred to as new amplification reagent packs. Amplification
reagent packs that have been used once and contain prepared master mix are referred to as partial amplification reagent packs. Refer to the
instructions provided in this manual for additional details.

Storage Conditions (Amplification Reagent Pack and Internal Control Vials)

Pack Type Storage Temperature Storage Time
New Packs -25 to -15°C Until date shown on label
New IC -25 to -15°C Until date shown on label
Partial Packs -25 to -15°C (protected from light) Up to 7 days after initial use
Partial IC -25 to -15°C Up to 14 days after initial use

Select combination of new and/or partial

packs required for run.
New Pack(s) Partial Pack(s)
Remove new pack(s) from
-15°C freezer (–25 to –15°C).
-25°C Thaw at 2 to 8°C or
15 to 30°C.

Once thawed, store at

2 to 8°C for ≤ 24 hours,
prior to use.

Complete sample extraction.

Remove caps. Remove partial pack(s) -15°C
from freezer (–25 to
–15°C). Confirm that -25°C
master mix is thawed
before use.

Load pack(s):
Firmly seat all packs;
partial packs to left.
≤ 60 minutes

≤ 25 minutes
Load master mix tube
Initialize Abbott if using more than 1 pack.
m2000rt.

Start master mix protocol.

Master mix protocol complete.

Seal PCR plate.

≤ 50 minutes ≤ 50 minutes
Recap partial pack(s) Transfer PCR plate
after first use. to Abbott m2000rt.

-15°C
Store partial pack(s) at –25 to
–15°C, upright and protected Start Abbott m2000rt
-25°C
from light. protocol.
Discard empty packs
and partial packs after
second use.

• Amplification reagent packs eligible for extended use must have a 6-digit serial number above the barcode.
• Partial amplification reagent packs can only be used a second time on the same instrument as the initial use. Using them on a different
instrument will generate a processing error, which may delay the run.
• Partial and new amplification reagent packs may be used together. All amplification reagent packs used on the instrument for a run must have
the same lot number.

20
APPENDIX 2. OPTIONAL UNG PROCEDURE FOR • If performing 25 to 48 reactions, prepare a second amplification
PROTOCOLS I, II, AND III master mix with a second Amplification Reagent Pack.
The uracil-N-glycosylase (UNG) procedure is to be used in conjunction NOTE: The Abbott m2000rt protocol (step 20) must be initiated
with the Abbott RealTime HIV-1 assay as an optional contamination within 50 minutes of the addition of Activation Reagent
control for customer laboratories that are currently using or have into the first rTth Enzyme Reagent bottle (step 13). This
previously used amplification technologies that incorporate uracil into 50 minutes includes 10 minutes incubation at room
the amplification product. temperature (step 19, below).
14. Pipette the contents of the master mix from the enzyme bottle(s)
REAGENTS into a single-use RNase/DNase-free tube and vortex to mix.
Uracil-N-glycosylase (UNG), List No. 06L87-02 (1 tube, 112 µL, 1U/µL)
15. Place an Abbott 96-Well Optical Reaction Plate in a StrataCooler
Description 96 or Eppendorf PCR Cooler stored as indicated in the instruction
Uracil DNA glycosylase (uracil-N-glycosylase) removes uracil residues manual. Using a DEDICATED PIPETTE, dispense 50-μL aliquots of
from the sugar moiety of single- and double-stranded DNA without the amplification master mix into the Abbott 96-Well Optical Reaction
destroying the phosphodiester backbone, preventing its use as a Plate. A calibrated repeat pipettor may be used. Visually verify that
hybridization target or as a template for DNA polymerases. Uracil DNA 50 µL has been dispensed into each well.
glycosylase will not remove uracil from RNA. 16. Transfer the Abbott 96-Well Optical Reaction Plate on the
Active Ingredients StrataCooler 96 or Eppendorf PCR Cooler to the Sample Preparation
• Uracil-N-glycosylase (UNG; < 0.1%) Area.
• Tween 20 (< 0.1%) Sample Preparation Area
Storage and Handling 17. In the Sample Preparation Area, transfer 50 μL of sample eluate to
The product is shipped on dry ice. the Abbott 96-Well Optical Reaction Plate on the StrataCooler 96 or
-15°C Eppendorf PCR Cooler. Use a separate pipette tip for each sample
Store at –25° to –15°C. eluate transfer. During the transfer of each sample, mix the reaction
-25°C by pipetting up and down 3 to 5 times. Visually verify that 100 µL has
been dispensed into each well.
UNG Limited License 18. Seal the Abbott 96-Well Optical Reaction Plate according to the
This product is sold under licensing arrangements between Celera instructions in the Abbott m2000rt Operations Manual.
Corporation and Invitrogen Corporation. The purchase price of this
product includes limited, nontransferable rights under US Patents 19. Remove the Abbott 96-Well Optical Reaction Plate from the
5,035,996; 5,683,896; 5,945,313; and 6,287,823 and foreign equivalents StrataCooler 96 or Eppendorf PCR Cooler to the Abbott Splash-
owned by Invitrogen Corporation to use only this amount of the Free Support Base. Centrifuge the Abbott 96-Well Optical
product to practice the claims in said patents solely for activities of the Reaction Plate in the Abbott Splash-Free Support Base at 5000g
purchaser. Further information on purchasing licenses under the above for 5 minutes. Incubate at room temperature (15 to 30°C) for 10
patents may be obtained by contacting Licensing Department, Invitrogen minutes. Centrifugation may take place during the 10-minute room
Corporation, 1600 Faraday Avenue, Carlsbad, CA 92008. temperature incubation. Following room temperature incubation,
transfer the Abbott 96‑Well Optical Reaction Plate on the Abbott
OPTIONAL UNG PROCEDURE FOR ASSAY Splash-Free Support Base to the Amplification Area.
PROTOCOL I: USING ABBOTT m1000 SYSTEM OR NOTE: Do not transfer the StrataCooler 96 or Eppendorf PCR Cooler
THE MANUAL SAMPLE PREPARATION METHOD AND to the Amplification Area.
ABBOTT m2000rt INSTRUMENT Amplification Area
NOTE: The step numbering from Protocol I is maintained. Starting 20. Place the Abbott 96-Well Optical Reaction Plate in the Abbott
from Step 11, execute the following: m2000rt instrument. From the Protocol screen, select the appropriate
Amplification Area application file corresponding to the sample volume being tested.
11. Switch on and initialize the Abbott m2000rt instrument. Initiate the Abbott RealTime HIV-1 protocol as described in the Abbott
NOTE: The Abbott m2000rt instrument requires 15 minutes to m2000rt Operations Manual, Operating Instructions section.
warm up.
ASSAY PROTOCOL II: OPTIONAL UNG PROCEDURE
12. Create the Abbott m2000rt test order. Refer to the Operating
Instructions section of the Abbott m2000rt Operations Manual. WITH PLASMA SAMPLES PREPARED FOR
From the Protocol screen, select the appropriate application file AMPLIFICATION USING THE ABBOTT m2000sp
corresponding to the sample volume being tested. NOTE: The step numbering from Protocol II is maintained. Starting
• Enter calibrator (needed if a calibration curve has not been stored from Step 11, execute the following:
on the Abbott m2000rt) and control lot-specific values in the test The Abbott m2000sp Master Mix Addition protocol (step 12) must be
order for accurate calibration and control evaluation. Lot-specific initiated within 1 hour after completion of Sample Preparation.
values are specified in each Abbott RealTime HIV-1 Calibrator and NOTE: Change gloves before handling the amplification reagents.
Control Kit Card. 11. Load the amplification reagents and the master mix tube (if needed)
Reagent Preparation Area on the Abbott m2000sp worktable after sample preparation is
completed. The following table shows the number of amplification
All reagent preparation must take place in the dedicated Reagent reagent packs needed based on the number of reactions.
Preparation Area. Refer to the Handling Precautions section of the If only 1 amplification reagent pack is being used, no master mix
package insert before preparing reagents. tube is required.
NOTE: Change gloves before handling the amplification reagents.
13. Prepare the amplification master mix. Amplification Reagent Pack Requirements a
• Each Amplification Reagent Pack supports up to 24 reactions. 1 to 24 25 to 48 49 to 72 73 to 96
• Prior to opening the amplification reagents, ensure that the Reactions Reactions Reactions Reactions
contents of the vials are at the bottom by tapping the vials in an 1 if new; 2 if new; 3 if new; 4 new
upright position 5 to 10 times on the bench to bring the liquid to up to 4 with up to 4 with up to 4 with or
the bottom of the vials. partial packs partial packs partial packs partial packs
• Use a PIPETTE DEDICATED FOR REAGENT USE ONLY to add a Refer to the Abbott m2000sp Operations Manual (List No. 9K20-06 or
27 µL of UNG to the Thermostable rTth Polymerase Enzyme higher) for instructions on inventory management to determine the maximum
bottle (Reagent 3). number of reactions that can be tested with the partial packs selected.
• Prepare the master mix by using a PIPETTE DEDICATED FOR
REAGENT USE ONLY to add 271 μL of the Activation Reagent
(Reagent 1) and 949 μL of the HIV-1 Oligonucleotide Reagent
(Reagent 2) together in the Thermostable rTth Polymerase
Enzyme bottle (Reagent 3).

21
 • Partial amplification reagent packs can only be used on the same Volume of UNG to Add to the Reagent Vial in
Abbott m2000sp instrument used for the reagent pack’s initial Position 3 of Each Amplification Reagent Pack
preparation. Using an amplification reagent pack for a second Tests Remaining in Add This Volume of
time on a different instrument will result in an error, which may the Pack UNG (μL)
delay the run.
1 5
• Partial and new amplification reagent packs may be
used together. 2 6
3 7
IMPORTANT: Partial amplification reagent packs should be stored
at – 25 to – 15°C until immediately before the second use. 4 8
Confirm that master mix is thawed before placing partial 5 9
pack(s) on the Abbott m2000sp worktable. Once removed from 6 10
– 25 to – 15°C, partial amplification reagent packs being used 7 11
a second time must be used within 25 minutes or discarded. 8 12
This applies to cumulative room temperature exposure, 9 13
including instances where packs are removed from storage, 10 13
but not used. 11 14
• Ensure that the contents of new amplification reagent packs 12 15
are at the bottom of the vials prior to opening the amplification
13 16
reagents by tapping the vials in an upright position on the bench
5 to 10 times. 14 17
• Do not tap partial amplification reagent packs being used a 15 18
second time. Tapping may result in loss of master mix volume in 16 19
the cap. 17 20
• Remove caps. If a new amplification reagent pack will be stored 18 21
for a second use, the vials will need to be recapped for storage. 19 22
If planning to reuse the original caps to recap the reagent vials, 20 23
save the original caps. If planning to use fresh caps to recap the 21 24
reagent vials, original caps may be discarded. 22 25
• Use a PIPETTE DEDICATED FOR REAGENT USE ONLY to add 23 26
specified volume of 1 U/µL UNG (List No. 06L87-02) to the 24 (new pack) 27
reagent vial in position 3 of new and partial amplification reagent
packs. 12. Select the appropriate deep well plate that matches the
• Use the table below to determine the volume of UNG to add to the corresponding sample preparation extraction. Initiate the Abbott
reagent vial in position 3 of new and partial amplification reagent m2000sp Master Mix Addition protocol. Follow the instructions as
packs. The reagent vial in position 3 of a new reagent pack described in the Abbott m2000sp Operations Manual, Operating
contains the Thermostable rTth polymerase enzyme. The reagent Instructions section.
vial in position 3 of a partial reagent pack contains master mix. NOTE: The operator should not manually fill any empty/unfilled
NOTE: The volume of UNG added to the reagent vial in position 3 wells in the Abbott 96-Well Optical Reaction Plate.
depends upon the number of tests remaining in the reagent • After sample extraction is complete, the Abbott m2000sp
pack, and not the number of samples being run. Refer to the automatically fills any empty wells in the Abbott 96-Well Optical
Abbott m2000sp Operations Manual for instructions pertaining Reaction Plate when there are greater than 48 samples processed
to amplification reagent pack inventory management and how within a run. Plate fill is not performed for runs containing 48
to determine the number of tests remaining in a reagent pack. samples or fewer.
• Manually mix by pipetting gently up and down for all partial • If prompted by the instrument, Reagent Carrier 2 should remain
packs. Do not mix new packs. in place, minimally containing the reagent vessel for mElution
• Partial amplification packs are loaded to the left of new Buffer (Reagent Carrier 2, location 6). If this reagent vessel has
amplification reagent packs on the Abbott m2000sp worktable. been unloaded, place a new reagent vessel with the mElution
• Ensure that amplification reagent packs are firmly seated on Buffer label into Reagent Carrier 2, location 6. System fluid will
the instrument. be added to the reagent vessel and used to fill empty wells. Once
this process is complete, the system will continue with the master
mix addition.
NOTE: System instructions for use of the automated plate-filling
feature are found in the Abbott m2000sp Operations
Manual (List No. 9K20 version 6 or higher), section 5,
Operating Instructions, Sample Extraction – Closed Mode.
• The Abbott m2000rt protocol (step 16) must be started within
50 minutes of the initiation of the Master Mix Addition protocol
(step 12).
NOTE: If the run is aborted for any reason subsequent to step 12,
a new 96-well Optical Reaction Plate must be used if the
Abbott m2000sp Master Mix Addition Protocol (step 12)
will be repeated.
13. Switch on and initialize the Abbott m2000rt instrument in the
amplification area.
NOTE: The Abbott m2000rt requires 15 minutes to warm-up.
NOTE: Remove gloves before returning to the sample preparation
area.
14. Seal the Abbott 96-Well Optical Reaction Plate after the Abbott
m2000sp instrument has completed addition of samples and master
mix according to the Abbott m2000sp Operations Manual, Operating
Instructions section.
15. Keep the sealed optical reaction plate to the Abbott Splash-Free
Support Base and incubate at room temperature (15 to 30°C) for
10 minutes. Following room temperature incubation, transfer the
Abbott 96-Well Optical Reaction Plate on the Abbott Splash-Free
Support Base to the Abbott m2000rt instrument.

22
16. Place the Abbott 96-Well Optical Reaction Plate in the Abbott • Use a PIPETTE DEDICATED FOR REAGENT USE ONLY to add
m2000rt instrument. From the Protocol screen, select the appropriate specified volume of 1 U/µL UNG (List No. 06L87-02) to the
application file corresponding to the sample volume being tested. reagent vial in position 3 of new and partial amplification reagent
Initiate the Abbott RealTime HIV-1 protocol as described in the Abbott packs.
m2000rt Operations Manual, Operating Instructions section. • Use the table below to determine the volume of UNG to add to the
NOTE: Test order transfer through the use of CD-ROM or network reagent vial in position 3 of new and partial amplification reagent
connection with export and import features of the m2000sp packs. The reagent vial in position 3 of a new reagent pack
and m2000rt software is recommended. If creating the contains the Thermostable rTth polymerase enzyme. The reagent
Abbott m2000rt test order manually, enter sample IDs in the vial in position 3 of a partial reagent pack contains master mix.
corresponding PCR tray locations according to the “Wells NOTE: The volume of UNG added to the reagent vial in position 3
for Selected Plate” grid, found on the detail screen of the depends upon the number of tests remaining in the reagent
“PCR Plate Results” on the Abbott m2000sp. See Section pack, and not the number of samples being run. Refer to
5 of the Abbott m2000sp Operations Manual. the Abbott m2000sp Operations Manual for instructions
17. If a prepared partial amplification reagent pack is to be used a pertaining to amplification reagent pack inventory
second time, cap the 3 reagent vials with the saved caps or new management and how to determine the number of tests
caps (List No. 3N20-01) and promptly store the reagents at – 25 to remaining in a reagent pack.
– 15°C, protected from light, and in an upright position. Discard any • Manually mix by pipetting gently up and down for all partial
amplification reagent packs that are exhausted or have been packs. Do not mix new packs.
used twice. • Partial amplification packs are loaded to the left of new
IMPORTANT: Amplification reagents that will be used a second time amplification reagent packs on the Abbott m2000sp worktable.
must be stored at – 25 to – 15°C within 50 minutes of the • Ensure that amplification reagent packs are firmly seated on
initiation of the master mix addition protocol. the instrument.
ASSAY PROTOCOL III: OPTIONAL UNG PROCEDURE Volume of UNG to Add to the Reagent Vial in
WITH DBS SAMPLES PREPARED FOR AMPLIFICATION Position 3 of Each Amplification Reagent Pack
USING THE ABBOTT m2000sp Tests Remaining in Add This Volume of
NOTE: The step numbering from Protocol III is maintained. Starting the Pack UNG (μL)
from Step 22, execute the following: 1 5
The Abbott m2000sp Master Mix Addition protocol (step 23) must be 2 6
initiated within 1 hour after completion of Sample Preparation. 3 7
NOTE: Change gloves before handling the amplification reagents. 4 8
22. Load the amplification reagents and the master mix tube (if needed) 5 9
on the Abbott m2000sp worktable after sample preparation is 6 10
completed. The following table shows the number of amplification 7 11
reagent packs needed based on the number of reactions. 8 12
If only 1 amplification reagent pack is being used, no master mix
tube is required. 9 13
10 13
Amplification Reagent Pack Requirements a 11 14
1 to 24 25 to 48 49 to 72 73 to 96 12 15
Reactions Reactions Reactions Reactions 13 16
14 17
1 if new; 2 if new; 3 if new; 4 new
up to 4 with up to 4 with up to 4 with or 15 18
partial packs partial packs partial packs partial packs 16 19
a Refer to the Abbott m2000sp Operations Manual (List No. 9K20 version 6 or
17 20
higher) for instructions on inventory management to determine the maximum 18 21
number of reactions that can be tested with the partial packs selected. 19 22
• Partial amplification reagent packs can only be used on the same 20 23
Abbott m2000sp instrument used for the reagent pack’s initial 21 24
preparation. Using an amplification reagent pack for a second 22 25
time on a different instrument will result in an error, which may 23 26
delay the run. 24 (new pack) 27
• Partial and new amplification reagent packs may be
used together. 23. Select the appropriate deep well plate that matches the
IMPORTANT: Partial amplification reagent packs should be stored corresponding sample preparation extraction. Initiate the Abbott
at – 25 to – 15°C until immediately before the second use. m2000sp Master Mix Addition protocol. Follow the instructions as
Confirm that master mix is thawed before placing partial described in the Abbott m2000sp Operations Manual, Operating
pack(s) on the Abbott m2000sp worktable. Once removed from Instructions section.
– 25 to – 15°C, partial amplification reagent packs being used NOTE: The operator should not manually fill any empty/unfilled
a second time must be used within 25 minutes or discarded. wells in the Abbott 96-Well Optical Reaction Plate.
This applies to cumulative room temperature exposure, • After sample extraction is complete, the Abbott m2000sp
including instances where packs are removed from storage, automatically fills any empty wells in the Abbott 96-Well Optical
but not used. Reaction Plate with mElution buffer when there are greater than
• Ensure that the contents of new amplification reagent packs 48 samples processed within a run. Plate fill is not performed for
are at the bottom of the vials prior to opening the amplification runs containing 48 samples or fewer.
reagents by tapping the vials in an upright position on the bench • If prompted by the instrument, Reagent Carrier 2 should remain
5 to 10 times. in place, minimally containing the reagent vessel for mElution
• Do not tap partial amplification reagent packs being used a Buffer (Reagent Carrier 2, location 6). If this reagent vessel has
second time. Tapping may result in loss of master mix volume in been unloaded, place a new reagent vessel with the mElution
the cap. Buffer label into Reagent Carrier 2, location 6. System fluid will
• Remove caps. If a new amplification reagent pack will be stored be added to the reagent vessel and used to fill empty wells. Once
for a second use, the vials will need to be recapped for storage. this process is complete, the system will continue with the master
If planning to reuse the original caps to recap the reagent vials, mix addition.
save the original caps. If planning to use fresh caps to recap the
reagent vials, original caps may be discarded.

23
 NOTE: System instructions for use of the automated plate-filling
feature are found in the Abbott m2000sp Operations
Manual (List No. 9K20 version 6 or higher), section 5,
Operating Instructions, Sample Extraction – Closed Mode.
• The Abbott m2000rt protocol (step 27) must be started within
50 minutes of the initiation of the Master Mix Addition protocol
(step 23).
NOTE: If the run is aborted for any reason subsequent to step 23,
a new 96-well Optical Reaction Plate must be used if the
Abbott m2000sp Master Mix Addition Protocol (step 23)
will be repeated.
24. Switch on and initialize the Abbott m2000rt instrument in the
amplification area.
NOTE: The Abbott m2000rt requires 15 minutes to warm-up.
NOTE: Remove gloves before returning to the sample preparation
area.
25. Seal the Abbott 96-Well Optical Reaction Plate after the Abbott
m2000sp instrument has completed addition of samples and master
mix according to the Abbott m2000sp Operations Manual, Operating
Instructions section.
26. Keep the sealed optical reaction plate to the Abbott Splash-Free
Support Base and incubate at room temperature (15 to 30°C) for
10 minutes. Following room temperature incubation, transfer the
Abbott 96-Well Optical Reaction Plate on the Abbott Splash-Free
Support Base to the Abbott m2000rt instrument.
27. Place the Abbott 96-Well Optical Reaction Plate in the Abbott
m2000rt instrument. From the Protocol screen, select the HIV-1 DBS
Viral Load application file. Initiate the Abbott RealTime HIV-1 protocol
as described in the Abbott m2000rt Operations Manual, Operating
Instructions section.
NOTE: Test order transfer through the use of CD-ROM or network
connection with export and import features of the m2000sp
and m2000rt software is recommended. If creating the
Abbott m2000rt test order manually, enter sample IDs in the
corresponding PCR tray locations according to the “Wells
for Selected Plate” grid, found on the detail screen of the
“PCR Plate Results” on the Abbott m2000sp. See Section
5 of the Abbott m2000sp Operations Manual.
28. If a prepared partial amplification reagent pack is to be used a
second time, cap the 3 reagent vials with the saved caps or new
caps (List No. 3N20-01) and promptly store the reagents at – 25 to
– 15°C, protected from light, and in an upright position. Discard any
amplification reagent packs that are exhausted or have been
used twice.
IMPORTANT: Amplification reagents that will be used a second time
must be stored at – 25 to – 15°C within 50 minutes of the
initiation of the master mix addition protocol.

24