Whole blood, serum or plasma samples may be

A qualitatitve, visually read in vitro immunoassay
for the detection of antibodies to Human Immunodeficiency Virus Type 1 (HIV-1) and type 2 (HIV-2) in
human whole blood (venous or fingerstick), serum
or plasma specimens.
NAME AND INTENDED USE
The Calypte® AwareTM HIV-1/2 BSP test is a single
use, qualitative, visually read, in vitro immunoassay
for the detection of antibodies to Human
Immunodeficiency Virus Type 1 (HIV-1) and Type 2
(HIV-2) in human whole blood (venous or finger-stick),
serum, or plasma specimens. It is intended for use as
a point-of-care aid in the clinical diagnosis of HIV
infection. This test may be used as a component of a
multi-test rapid algorithm in conjunction with other
approved HIV antibody assays.
SUMMARY AND EXPLANATION OF THE TEST
Acquired Immune Deficiency Syndrome (AIDS), AIDS
related complex (ARC), and pre-AIDS are thought to
be caused by the Human Immunodeficiency Viruses
(HIV) Type 1 and Type 21-4. Individuals infected with
HIV produce antibodies against the HIV viral proteins.
Testing for the presence of these antibodies in bodily
fluids (e.g., blood, oral fluid, urine) is an accurate aid in
the diagnosis of HIV infection5-9. The Calypte®
AwareTM HIV-1/2 BSP test is a point-of-care test to aid
in the diagnosis of infection with HIV-1 or HIV-2.
The test results are interpreted after 20 minutes but not
more than 45 minutes after the introduction of the
assay test strip to the diluted specimen.
8. Use adequate lighting to visually check a test
result. If two lines are present at any visible
intensity, the test result is interpreted as reactive
(see Interpretation of Results section).
MATERIALS PROVIDED
Each Calypte® Aware™ HIV-1/2 BSP Test (25 or 50
test kit) contains:
• One package insert (this document)
• 25 or 50 Foil Pouches. Each pouch contains one
device (assay test strip) and a desiccant
• 2 dropper bottles of Blood Sample Buffer
• (5 mL each)
• 1 or 2 packages of Specimen Dilution Tubes
(25 each)
• 1 or 2 packages of Specimen Collection Loops
(25 each)
6. Use each test device (assay test strip) only once.
If a test must be repeated, start the process over
using a new test device.
7. Do not use the test beyond the expiration date
printed on the foil pouch or Sample Buffer.
TEST STORAGE
Store unused Calypte® Aware™ HIV-1/2 BSP pouches
and Sample Buffer unopened at 2-30˚C (36-86˚F). Do
not open the foil pouch containing the assay test strip
until you are ready to perform a test. If stored
refrigerated or below room temperature, allow Blood
Sample Buffer and unopened pouched device to come
to room temperature (15-30˚C or 59-86˚F) before use.
TEST PROCEDURE
1. Mix the Blood Sample Buffer by gently inverting the
bottle about 3 times.
2. Remove the cap from the bottle, aspirate buffer to
the fill line and dispense it into a clean Specimen
Dilution Tube.
Also available separately are:
Calypte® Aware™ HIV-1/2 BSP Controls.
2. Practice universal precautions10-13 when handling
whole blood, serum, or plasma specimens, and
used assay test strips or tubes.
3. Do not drink, eat or smoke in areas where
specimens are being handled.
4. Wear gloves, a lab coat, and eye protection when
handling specimens or devices that have come into
contact with blood or blood components. Wash
hands thoroughly after performing each test. Dispose
of gloves in a biohazard waste container after use.
Visually inspect the loop to
make sure that it is completely
filled with blood, serum or plasma.
4. Immediately place the loop containing the sample
into the tube containing the Blood Sample Buffer.
Swirl the loop in the tube to mix thoroughly.
5. Remove the sample loop and discard into
biohazard waste.
6. Tear open the foil pouch containing the assay test
strip. Tear the pouch at the location of the notch
on the side of the pouch. Remove the assay test
strip from the pouch. Avoid touching the membrane surface in the middle of the strip with your
fingers.
7. Place the assay test strip in the
tube containing the diluted
specimen, with the arrows on the
assay test strip pointing down.
3. Follow one of the two methods below that
corresponds to the specimen type to be tested.
Finger-stick blood sample14
Using an antiseptic wipe, clean the finger of
the person being tested. Allow the finger to
dry thoroughly or wipe dry with a sterile gauze
pad.
Using a sterile lancet, puncture
the skin just off the center of the
finger pad. Squeeze the finger
gently to obtain a drop of blood.
Avoid squeezing or milking the
finger to accelerate bleeding, as
this may dilute the blood with
excess tissue fluid.
Using the Specimen Collection
Loop, touch the round end of
the loop to the drop of blood.
Visually inspect the loop to
make sure that it is completely
filled with blood.
8. Set a timer for 20 minutes, or note
the time the assay test strip was
added to the sample and wait 20
minutes.
20
Read test result after 20 minutes.
Do not read the result more than
45 minutes after beginning the test.
Record the test result seen in the
Test and Control Zones of the assay
test strip. (Refer to Test Results and Interpretation and Limitations of the Procedure, below).
HIV 1&2
HIV 1&2
1. This test should be performed at ambient
temperature (15-30˚C or 59-86˚F).
Using the Specimen Collection
Loop provided, touch the round
end of the loop to the whole,
blood, separated serum or plasma.
HIV 1&2
HIV 1&2
WARNINGS AND PRECAUTIONS
Test is for in vitro diagnostic use only. Read the
package insert completely before using the product.
Follow the instructions carefully. Failure to follow
instructions may result in inaccurate test results.
Whole blood, serum or plasma samples may be
obtained by collection of blood by conventional
venipuncture methods and serum or plasma
separated from blood cells Plasma specimens
may be collected with collection tubes containing
EDTA, or ACD (acid/citrate/dextrose)
anticoagulants.
HIV 1&2 BLOOD
HIV 1&2 BLOOD
MATERIALS REQUIRED BUT NOT PROVIDED
• 20-45 minute timer or watch
• Sterile lancet or blood collection system
(1 required per test)
• Antiseptic wipe (1 required per sample)
• Sterile gauze pads (1 required per sample)
Disposable gloves
Biohazard disposal container
Whole blood, serum or plasma sample15-16
HIV 1&2 BLOOD
HIV 1&2 BLOOD
The assay test strip contains synthetic peptides
representing the immunodominant regions of the HIV-1
gp41 and HIV-2 gp36 transmembrane proteins and a
Protein A antibody-capture procedural control
immobilized onto the nitrocellulose membrane in the
Test Zone and the Control Zone, respectively. To
perform the assay, a loopful (5 μl) of blood, serum or
plasma specimen is added to approximately 200 μl of
Blood Sample Buffer in a test tube and mixed in the
test tube. The assay test strip is then placed vertically
into the test tube containing the sample/buffer mixture.
As the diluted specimen migrates up the assay test
strip, it rehydrates a reddish Protein A-colloidal gold
reagent (“conjugate”) on the strip and IgG in the
specimen becomes bound to the Protein A/colloidal
gold particles (“IgG/conjugate complex”). The
specimen/conjugate mixture continues to migrate up
the strip, and first encounters the Test Zone of the
assay test strip containing the HIV antigens. If the
specimen contains antibodies to HIV, the
IgG/conjugate complex binds to the antigen and
becomes immobilized at the antigen line in the Test
Zone and a reddish colored line appears. This
indicates a reactive result. The intensity of the line is
not proportional to the amount of antibody present in
the specimen. The absence of a colored line in the
WARNINGS AND PRECAUTIONS (Continued)
5. Dispose of all test specimens and materials used
in the test procedure in a biohazard waste
container. Lancets should be placed in a
puncture-resistant container prior to disposal. Used
materials may be incinerated. Liquid wastes may
be mixed with appropriate chemical disinfectants.
A solution of 10% bleach (0.5% solution of sodium
hypochlorite) is recommended. Allow 60 minutes
for effective decontamination.
HIV 1&2 BLOOD
HIV 1&2 BLOOD
BIOLOGICAL PRINCIPLES OF THE TEST
The Calypte® AwareTM HIV-1/2 BSP test is a manually
performed, visually read, 20 minute qualitative (i.e.
“yes/no”) immunochromatographic assay for the
detection of antibodies to HIV-1 and 2 in venous or
finger-stick whole blood, serum, or plasma. The
Calypte® AwareTM HIV-1/2 BSP test is composed of a
single-use test device and a bottle of buffered diluent
for the blood, serum or plasma. The test utilizes a
proprietary lateral flow immunoassay procedure. The
assay test strip is comprised of several materials that
provide the matrix for the immunocheromatography of
the specimen and the platform for the indication of the
test results.
Test Zone indicates that the specimen does not contain
anti-HIV antibodies. The specimen/conjugate mixture
continues to migrate up the assay test strip until it
encounters the Control Zone. The Control Zone
contains Protein A immobilized in a line on the assay
test strip. The remaining IgG/conjugate complexes
become bound to the Protein A and a reddish colored
line appears. The appearance of the control line is
evidence that the test functioned properly and
contained IgG. A reddish-purple control line will appear
in the Control Zone during the performance of all valid
tests, whether or not the sample is reactive or negative
for antibodies to HIV-1 or 2. The specimen continues
to migrate past the Control Zone into the final absorbent pad, which helps draw the specimen/conjugate
mixture through the strip and clear any background
color.
9. Dispose of the assay test strip and
tube in a biohazardous waste
container.
QUALITY CONTROL
A control line in the Control Zone indicates a valid
result. A valid result indicates a suitable sample was
collected and the test functioned properly. The
control line will appear on all valid tests, whether or
not the result is reactive. (Refer to Test Results and
Interpretation, below).
Calypte® Aware™ HIV-1/2 BSP Controls are
provided separately. The controls are used to verify
proper test performance. Both Positive and Negative
Controls should be run by each operator at the
beginning of each shift during which an unknown
specimen is tested. The Positive Control should be
run to provide an alignment guide to indicate the
location of the Control and Test Zones on the assay
test strip. Controls should also be run whenever
changing to a new operator, to a different lot of tests,
or when testing conditions (e.g. new location,
lighting, temperature etc.) change.
TEST RESULTS AND INTERPRETATION
View the Test and Control Zones of the assay test
strip as described below and shown in the diagram
below.
REACTIVE - two lines appear. If both a test and
control line appear, (i.e. two lines appear on the test
strip, in the Test Zone and Control Zone, respectively) the result is considered reactive. One of these
lines may be darker than the other.
At the right are examples of reactive
results. A reactive result means
that anti-HIV-1 or 2 antibodies have
been detected in the specimen.
This test result is interpreted as a
preliminary positive for HIV antibodies.
Note: using the Calypte® Aware™ HIV-1/2 BSP
Positive Control in a valid test gives this result.
NON-REACTIVE - only the control line appears. If a
single line appears on the test strip in the Control
Zone, the result is non-reactive.
The diagram at the right shows
examples of non-reactive results.
A non-reactive result suggests
the absence of reactive anti-HIV
antibodies in the specimen.
Note: using the Calypte® Aware™ HIV-1/2 BSP
Negative Control in a valid test gives this result.
INVALID - no control line present. If there is no
control line in the Control Zone, the
result is invalid (examples at right).
The result is invalid even if a test
line appears in the Test Zone. An
invalid test should be repeated with
a new test device or specimen.
LIMITATIONS OF THE PROCEDURE
1. The Calypte® Aware™ HIV-1/2 BSP test must be
used in accordance with these instructions to
obtain an accurate result.
2. Reading test results sooner than 20 minutes or
later than 45 minutes after the start of the test
may give erroneous results.
3. Blood, serum and plasma specimens for antibody
testing must be collected as detailed in the
procedure.
4. Plasma specimens collected in the presence of
an anti-coagulant other than EDTA or ACD have
not been evaluated.
5. Blood, plasma or serum specimens that have
been heat or chemically inactivated, or have
repeatedly been frozen and thawed, may not give
accurate results.
6. Whole blood specimens that are highly hemolyzed or are more than 48 hours old may give
erroneous results.
7. The test is not for use with body fluids other than
blood, plasma or serum collected as described in
the instructions above.
8. The intensity of the test line in a reactive result
does not necessarily correlate with the amount of
anti-HIV antibody in the specimen.
9. If a red background makes it difficult to read the
test at 20 minutes, wait until the background
clears to read the result (but not more than 45
minutes after starting the test).
Copyright 2007 Calypte Biomedical Corporation.
All Rights Reserved.
LN 20529.01
PERFORMANCE CHARACTERISTICS
Clinical Performance
Studies were conducted at three sites.
Performance on Standardized Specimen Panels and Clinical Specimens
Site 1 (Thailand)
A total of 1023 subjects presenting at an anonymous AIDS clinic were tested using the Calypte® Aware™
HIV-1/2 BSP assay. The Reference method was the Orgenics Rapid HIV-1/2 Blood Test, Doublecheck™ II
for initial screening. Reactive specimens were confirmed using the Bio-Rad GenScreen HIV-1/2 Version 2
ELISA and/or the Fujirebio Serodia®-HIV (HIV-1 only) Particle Agglutination Test. In this study, specificity was
100% (631/631) and sensitivity was 100% (392/392).
Performance on Standardized Specimen Panels
To demonstrate the performance of the Calypte® Aware™ HIV-1/2 test in comparison to other available tests, a series of standardized specimen panels were run
Licensed EIAs (signal to cutoff)
TM
Licensed EIAs (signal to cutoff)
Genetic Genetic Organon
Systems Systems Teknika
HIV-1
HIV-1/2
HIV-1
ID
Status
Aware
BSP
HIV-1/2
1
Pos
Pos
10.4
3.9
1.4
Pos
Pos
2
Neg
Neg
0.2
0.2
0.3
Neg
Neg
3
Pos
Pos
9.3
3.2
1.8
Pos
Pos
Neg
Abbott
HIV-1
Abbott
HIV-1/2
4
Pos
Pos
15.7
8.1
6.3
Pos
5
Pos
Pos
4.8
6.2
3.2
Pos
Pos
6
Pos
Pos
6.4
4.4
1.2
Pos
Neg
7
Pos
Pos
7.3
2.2
1.3
Pos
Pos
8
Pos
Pos
11.0
5.0
2.8
Pos
Pos
9
Pos
Pos
11.8
2.1
0.7
Pos
Neg
10
Pos
Pos
9.6
0.5
0.6
Pos
Neg
11
Pos
Pos
10.1
8.2
3.8
Pos
Pos
12
Pos
Pos
2.8
0.1
0.3
Pos
Neg
13
Pos
Neg
13.1
0.3
0.9
Pos
Pos
14
Pos
Neg
9.6
0.3
0.4
Neg
Neg
15
Pos
Number Correct
Pos
16.2
7.7
1.8
Pos
Neg
13/15
15/15
11/15
10/15
14/15
8/15
ID
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
Status
Pos
Pos
Pos
Pos
Neg
Pos
Pos
Pos
Pos
Pos
Pos
Pos
Pos
Pos
Pos
BBI Worldwide Anti-HIV Performance Panel WWRB302 (Modified)
Licensed EIAs (signal to cutoff)
ID
Genotype* (Origin)
Abbott
HIV-1
Abbott
HIV-1/2
Genetic Genetic Organon
Systems Systems Teknika
HIV-1
HIV-1/2
HIV-1
Genetic Genetic Organon
Systems Systems Teknika
HIV-1
HIV-1/2
HIV-1
AwareTM BSP
HIV-1/2
Neg
Pos
Neg
Pos
Neg
Pos
Pos
Neg
Neg
Pos
Pos
Pos
Neg
Pos
Pos
Abbott
HIV-1
Abbott
HIV-1/2
0.2
0.8
0.3
1.7
0.1
1.6
0.4
0.3
0.1
1.5
4.9
0.1
0.5
3.7
2.1
6.6
3.7
4.5
9.7
0.1
7.3
1.0
7.1
2.4
3.5
3.3
5.0
3.3
14.3
2.7
1.2
2.4
0.3
1.2
0.1
3.2
1.1
0.6
0.2
1.7
2.7
0.2
1.8
2.9
2.2
0.4
1.0
0.3
1.0
0.1
2.4
0.3
1.8
0.3
2.5
7.0
0.1
0.4
7.0
6.4
0.4
0.7
0.3
0.4
0.3
0.9
0.4
0.5
0.3
1.0
1.9
0.3
0.4
1.8
1.0
10/15
7/15
15/15
11/15
9/15
5/15
Number Correct
BBI HIV-1 Seroconversion Panels
Sample Type
AwareTM
BSP
HIV-1/2
AF (PRB-931)
AQ (PRB-941)
BB (PRB-952)
BG (PRB-957)
BH (PRB-958)
BH (PRB-959)
First Sequential Seroconverter Sample Detected (# days from first bleed)
Genetic
Genetic
Organon
Systems
Systems
Teknika
AwareTM BSP
Abbott
Abbott
HIV-1
HIV-1/2
HIV-1
HIV-1/2
HIV-1
HIV-1/2
15
21
17
28
17
14
33
21
17
n/a
n/a
n/a
28
18
14
28
15
9
35
21
14
28
17
14
33
25
>21
28
15
14
33
>25
21
>28
>17
14
1
O (Spain)
Pos
1.1
1.8
0.8
5.6
1.3
A (Ghana)
Pos
>11.5
>16.1
6.9
8.7
7.0
3
G (Ghana)
Pos
>11.5
>16.1
7.1
8.8
7.2
4
G (Ghana)
Pos
>11.5
>16.1
7.1
8.8
6.5
5
A (Ghana)
Pos
>11.5
>16.1
7.1
8.7
7.0
2. Curran JW, Morgan WM, Hardy AM, et al. The epidemiology of AIDS: current status and future prospects. Science 1985; 229:1352-7.
6
G (Ghana)
Pos
>11.5
>16.1
6.9
8.8
7.1
7
G (Ivory Coast)
Pos
>11.5
>16.1
6.9
8.7
6.7
3. Clavel F, Guetard D, Brun-Vezinet F, et al. Isolation of a new human retrovirus from West African patients with AIDS. Science 1986;
233:343-6.
8
A (Ivory Coast)
Pos
>11.5
>16.1
6.9
8.6
6.5
10
NEG (Ivory Coast)
Neg
0.4
0.2
0.1
0.4
0.4
11
HIV-2 (Mozambique)
Pos
1.2
14.6
0.6
9.7
3.0
12
C (Mozambique)
Pos
>11.5
>16.1
7.1
8.9
6.9
14
D (Uganda)
Pos
>11.5
>16.1
4.5
8.5
6.2
References
1. Gallo RC, Salahuddin SZ, Popovic M, et al. Frequent detection and isolation of cytopathic retroviruses (HTLV III) from patients with AIDS
and at risk for AIDS. Science 1984; 224:500-3.
6. Cao Y, Hosein B, Borkowsky W, Marabile M, et al. Antibodies to human immunodeficiency virus Type 1 in urine specimens of HIV-1
seropositive individuals. AIDS Research and Human Retroviruses. 1989; 5(3):311-319.
D (Uganda)
Pos
>11.5
>16.1
6.3
8.1
7.2
Pos
>11.5
>16.1
7.0
8.8
6.9
8. Regan, KJ, Lile CC, Book GW, Devash Y, et al. 1990. Use of urine for HIV-1 antibody screening. The Lancet. 1990; 335:358-359.
17
D (Uganda)
Pos
>11.5
>16.1
6.8
9.8
7.0
19
C (Zimbabwe)
Pos
>11.5
>16.1
6.0
9.9
7.0
9. Berrios DC, Avins AL, Haynes-Stansad K, Eversley R, Woods WJ. Screening for human immunodeficiency virus antibody in urine. Arch.
Pathol. Lab. Med. 1995. February 1995.
21
B (China)
Pos
>11.5
>16.1
6.7
8.8
7.0
10. CDC. Recommendations for prevention of HIV transmission in health-care settings. MMWR 1987; 36(SU02):3S-18S.
22
E (Thailand)
Pos
>11.5
>16.1
7.3
9.8
7.0
11. CDC. Perspectives in disease prevention and health promotion update: universal precautions for prevention of transmission of human
immunodeficiency virus, hepatitis B virus, and other bloodborne pathogens in health-care settings. MMWR 1988; 37(24):377-388.
E (Thailand)
Pos
>11.5
>16.1
7.4
9.8
6.9
Pos
0.4
15.4
3.8
10
2.1
26
B (USA)
Pos
>11.5
>16.1
7.4
9.8
7.1
27
B/D (USA)
Pos
>11.5
>16.1
7.0
9.8
7.2
28
F (Argentina)
Pos
>11.5
>16.1
7.0
8.9
6.8
29
B (Argentina)
Pos
>11.5
>16.1
6.9
8.5
5.6
30
NEG (Argentina)
Neg
0.3
0.2
0.2
0.2
0.4
25/25
24/25
25/25
25/25
25/25
25/25
*Genotype based on sequencing by PCR
0
0
631
Site 2 (Thailand)
A total of 682 subjects were tested using the Calypte® Aware™ HIV-1/2 BSP assay. In this study, specificity
was 100% (626/626) and sensitivity was 100% (56/56). The Reference test was a screen with the Abbott
AxSYM® HIV-1/2 Gr O Reagent, with confirmation of reactive results using Fujirebio Serodia®-HIV (HIV-1
only) Particle Agglutination Test, and Abbott Determine® HIV-1/2.
Reference Tests
Positive
Negative
Calytpe® Aware™ HIV-1/2 BSP
Positive
56
0
Negative
0
626
Site 3 (People’s Republic of China)
A total of 1506 subjects from the People’s Republic of China were tested using the Calypte® Aware™ HIV-1/2
BSP assay. A total of 247 were known to be HIV antibody positive and 1090 at low risk or antibody negative.
The remaining subjects were representative of subjects of unknown status with non-HIV diseases or
conditions (e.g. cancer, HCV infection, pregnancy, and other conditions). The reference method was the
Vironostika Uniform II Plus O EIA and the Genelabs HIV Western Blot. In this study, specificity was 99.3%
(1250/1259) and sensitivity was 100% (247/247).
Reference Tests
Positive
Negative
Calytpe® Aware™ HIV-1/2 BSP
Positive
Negative
247
9
0
1250
A total of 3211 subjects were tested using the Calypte® Aware™ HIV-1/2 BSP assay and various reference
assays. In these studies, specificity was 99.8% (2507/2513) and sensitivity was 100% (695/695)
Reference Tests
Positive
Negative
Calytpe® Aware™ HIV-1/2 BSP
7. Connell JA, Parry JV, Mortimer PP, Duncan RJS, et al. Preliminary report: accurate assays for anti-HIV in urine. The Lancet. 1990;
335:1366-1369.
D (Uganda)
HIV-2 (India)
392
5. Schochetman G. and George JR., Editors. AIDS Testing Methodology and Management Issues. Springer-Verlag, New York City, N.Y. 1992.
15
24
Positive
Negative
4. Rosenberg ZF and Fauci AS. The immunopathogenesis of HIV infection. Frank J. Dixon, Editor. Advances in Immunology. 1989;
47:377-431.
16
25
Calytpe® Aware™ HIV-1/2 BSP
All Sites Combined
2
Number Correct
Reference Tests
Positive
Negative
BBI Anti-HIV Low Titer Performance Panel (Modified) PBR-107
BBI Anti-HIV Low Titer Performance Panel PBR-108
Positive
695
9
Negative
0
2507
EXPLANATION OF SYMBOLS
12. CDC. Guidelines for prevention of transmsission of human immunodefficiency virus and hepatitis B virus to healthcare and public-safety
workers. MMWR 1989; 38(S-6):3-37.
13. Sehulster LM, Hollinger FB, Dreesman GR, and Melnick JL. Immunological and biophysical alteration of hepatitis B virus antigens by
sodium hypochlorite disinfection. Appl Env Microbiol 1981; 42: 762-7.
14. NCCLS Document H4-A4. Procedures and devices for the collection of diagnostic blood specimens by skin puncture; Approved standard.
NCCLS. Wayne PA, September 1999.
15. NCCLS Document H3-A4. Procedures for the collection of the diagnostic blood specimens by venipuncture; Approved standard-fourth
edition. NCCLS, Wayne PA, June 1998.
16290 S.W. Upper Boones Ferry Rd.
Portland, OR USA 97224
Calypte internet website: http://www.calypte.com
16. NCCLS Document H18-A2. Procedures for the handling and processing of blood specimens; Approved guideline-second edition. NCCLS,
Wayne PA, October 1999.
Copyright 2007 Calypte Biomedical Corporation.
All Rights Reserved.
LN 20529.01 (EN) 4/07
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