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Chamber for incubation
Disposable latex gloves
Lab timer
Fluorescent microscope equipped with 495 nm
exciter filter and 515 nm barrier filter.
PrEcautIonS
1. All human source materials used for this product
have been tested and found to be negative (not
repeatedly reactive) for antibodies to Human Im-
munodeficiency Virus-1 (HIV-1), Human Immuno-
deficiency Virus-2 (HIV-2), hepatitis C virus (HCV),
and for hepatitis B surface antigen (HBsAg) by FDA
approved methods. However, no test method can
offer complete assurance that HIV-1, HIV-2, hepa-
titis C, hepatitis B, or other infectious agents are
absent. Thus, all kit materials should be handled in
the same manner as potentially infectious materials.
2. All patient samples should be handled at the Bio-
safety Level 2 as recommended for any potentially
infectious human serum or blood specimen in the
Centers for Disease Control/National Institutes of
Health Manual: Biosafety in Microbiological and
Biomedical Laboratories, 1984 Edition.
3. Dilution of the components or substitution of com-
ponents other than those provided in this system
may yield inconsistent results.
4. Sodium azide is used as a preservative. Sodium
azide may react with lead or copper plumbing and
form explosive metal azide salts. When disposing
of reagents, flush with ample volumes of tap water
to prevent potential residues in plumbing. Sodium
azide is a poison and may be toxic if ingested.
5. This kit is for in vitro diagnostic use.
6. In the event hemolyzed or lipemic sera must be
used, heat inactivate sera 30 minutes at 56°C for
optimal results. Microbially contaminated sera
should not be used.
7. It has been demonstrated that false positive ANA's
may occur due to C1q binding to DNA, indepen-
dent of the substrate used (30). To eliminate poten-
tial C1q interference, routinely heat inactivate all
patient sera at 56°C for 30 minutes prior to testing.
8. The titratable control serum is intended for use in
monitoring lot-to-lot and run-to-run reproducibility.
It is not intended as a measurement of overall
sensitivity or specificity of the assay.
9. Do not smoke, eat, or drink in areas where speci-
mens or kit reagents are handled.
10. Avoid splashing or generation of aerosols at all
times.
11. Incubation times and temperatures other than
those specified may give erroneous results.
12. Cross contamination of reagents or samples may
give false results.
13. Reusable glassware must be washed and thor-
oughly rinsed free of detergents prior to use. All
glassware must be clean and dry before use.
14. Bring all reagents, slides, and specimens to room
temperature (18-24°C) prior to use.
15. Wear disposable latex gloves when handling speci-
mens and reagents, and wash hands thoroughly
afterwards.
16. Microbial contamination of reagents or samples
may give false results.
17. Never pipette by mouth and avoid contact of
reagents and specimens with skin and mucous
membranes. If contact occurs, wash with a germi-
cidal soap and copious amounts of water.
SPEcImEn collEctIon
collection: Serum is the preferred specimen. Approxi-
mately 5 ml of whole blood should be collected asepti-
cally by venipuncture using a sterile vacuum collection
tube or other suitable collection system. Allow blood to
clot at room temperature (18-24°C). Serum should be
separated from the clot by centrifugation as soon as
possible to minimize hemolysis.
Interfering Substances: Sera exhibiting a high degree
of hemolysis, icterus, lipemia, or microbial growth
should not be used because these conditions may
cause aberrant results. Specimens containing visible
particulate matter should be clarified by centrifugation
before testing.
Storage: Sera may be stored at 2-10°C up to one week.
If testing is further delayed, sera should be stored frozen
at -20°C or lower. Serum should not be stored in a self-
defrosting refrigerator or freezer.
cautIon: Repeated freeze/thawing of patient samples
may yield false positive or false negative results.
IntErPrEtatIon oF rESultS
QualItY control
Positive, negative and PBS controls should be run in
the wells provided for quality control on each slide.
The positive control should show bright apple-green
fluorescence in the nuclei of the cells, with a clearly
discernible pattern characteristic of the control serum
that was used. The negative control should show low
intensity, nonspecific dull green fluorescence in both
the cytoplasm and nucleus, but with no discernible
pattern of nuclear staining. The PBS control is used to
observe non-specific staining by the antibody reagent,
and should not exhibit any green fluorescence. If the
controls do not appear as described, the test is invalid
and should be repeated.
oPtIonal tItrataBlE control
When reading titers, many laboratories begin reading
with the well that contains the most dilute sample and
read "backwards" to the 1:40 dilution. The first well in
which a clearly discernible nuclear staining pattern is
visible is the titer end-point. We recommend this tech-
nique for determining titer end-points.
The mean titer and titer range (± one dilution on either
side of the mean) determined for this lot number were
established in our laboratory and are stated as a guide.
This control is provided to allow each laboratory to
assess the reproducibility (precision) of its ANA testing.
Since this control is not intended to be an indicator
of titer accuracy, each laboratory should establish its
own mean titer end-point for this sample, and should
use this information to assess run-to-run reproducibility
(precision).
Through multiple testing of this titratable control, using
the Immuno Concepts Fluorescent ANA Test System,
a mean titer value has been established for each lot
number. The lot number, mean titer and titer range (±
one twofold dilution on either side of the mean) are
stated on the vial label and should be used as a guide
for the test system performance.
It is important that the intensity of fluorescence not be
confused with the presence or absence of antinuclear
antibodies. The key factor to consider in determining
whether a given dilution of serum is positive is the ap-
pearance of a clearly discernible pattern, irrespective
of the intensity of the fluorescent staining.
This titratable control will show the typical speckled
pattern associated with the RNP antibody. Also present
may be a second pattern of NSp I (several discrete
speckles in the nucleus of interphase cells), however, it
is the typical RNP speckled pattern that is to be used for
the purpose of reading end-point.
The values obtained in our laboratory may differ from
your values. Some of the many factors that can affect
your results may include, but are not limited to:
1. The type of light source used. Mercury light sources
will produce greater excitation energy at 495
nm than Quartz/Halogen. The 50-watt, 100-watt,
and 200-watt Mercury light sources differ little
in excitiation energy at 495 nm. The 100-watt
Quartz/Halogen light sources will produce greater
excitation energy at 495 nm than 50-watt Quartz/
Halogen.
2. The condition and age of the light source. This is
particularly true for Mercury light sources which
generally exhibit a gradual reduction in excitation
energy at 495 nm prior to burning out. This gradual
reduction in excitation energy can result in a
significant loss in sensitivity over several weeks. This
problem can be avoided by keeping a time log.
For best results, replace 50-watt mercury bulbs at
100 hours, and 100 or 200-watt mercury bulbs at 200
hours. Quartz/Halogen light sources generally do
not exhibit a gradual reduction in excitation energy
prior to burning out.
3. The type of exciter filter used. Interference exciter
filters provide greater sensitivity over a much nar-
rower wavelength than absorption exciter filters.
Refer to your fluorescent microscope manual or
sales representative for more information.
4. Proper alignment of the microscope light path.
Refer to your fluorescent microscope manual for
instructions.
5. The numerical aperture of the objective. With
incident light fluorescence (Epi), fluorescence is
increased exponentially as the numerical aperture
(NA) of the objective is increased additively. This
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