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What
is the significance of a negative ANA and a positive dsDNA?
The positive antinuclear antibody (ANA) test
and the presence of autoantibodies to double stranded DNA
(dsDNA) were established as two of the possible eleven criteria
needed for the diagnosis of systemic lupus erythematosus (SLE)
(American College of Rheumatology,1982). For this reason it
has become an increasingly common occurrence for a physician
to order both an ANA and a dsDNA test at the same time. (1)
In the course of routine diagnostic work, several of our customers
have run across samples that give conflicting results: a
negative ANA and a positive dsDNA.
Ideally the ANA test should be run first. This allows the
slide to be used as an initial screen to indicate which samples
will require follow-up testing for the extractable nuclear
antigens (ENA) and/or dsDNA. Studies indicate that if the
ANA test is negative, “searching for . . . anti-dsDNA
is generally unjustifiable.” (2) Further support for
this statement can be found in an article published in CAP
Today, in which the author outlines his policy of not
performing Crithidia luciliae immunofluorescence (CLIF)
when the HEp-2 test is negative. He states that the ANA substrate
is highly sensitive for dsDNA and that CLIF “would not
be helpful in that situation.” (3) Unfortunately in many
labs the ANA and DNA tests are being run in parallel which
can lead to some confusing results.
The antibodies to dsDNA are very heterogeneous in regards
to their avidity, their isotype (class), and their ability
to cross-react. There are several options available when selecting
a test for the detection of dsDNA antibodies; however no one
test is both 100% specific and/or 100% sensitive. All of the
tests including the Farr assay, the enzyme linked immunosorbent
assays for dsDNA (dsDNA ELISA) and CLIF are a compromise between
specificity and sensitivity. Exact numbers vary from test
system to test system and from manufacturer to manufacturer,
but in general, ELISA is the most sensitive followed by the
Farr and Crithidia assays. However, ELISA is also the
least specific of the test systems—multiple studies have
shown positive dsDNA titers by ELISA in diseases other than
SLE. (4,7) Both the Farr and Crithidia assays have
been shown to be highly specific (ranging from 88-98%) for
SLE. (5,6,7)
All available systems have known causes of false positives.
High affinity antibodies of classes other than IgG (which
are of questionable clinical significance), and contamination
with C reactive protein, or single strand DNA (ssDNA) are
all potential causes of false positives in the Farr assay.
8 Antibodies to single stranded DNA or other constituents
used in manufacturing processes are known to give false positive
results in dsDNA ELISAs. (8) Lipoprotein-IgG complexes are
a major cause of false positive results in CLIF. (8,9) In
addition, antibodies to histone and other DNA related/associated
proteins (deoxyribonucleic proteins, DNPs) can result in false
positives for all of the test systems mentioned above. (8,10,11,12)
The clinical significance of a negative ANA in combination
with a positive dsDNA is unknown. While a negative ANA does
not conclusively rule out SLE, it should be noted that less
than 5% of known SLE patients have a negative ANA profile—making
SLE an unlikely diagnosis. (13) If the clinician insists that
SLE is still suspect, testing for anti-SSA/Ro may be advisable
as it can be missed by HEp-2 immunoflorescent assays. Many
ANA negative individuals have antibodies such as SSA/Ro that
can be detected in their blood by other more sensitive methods.
(13) In addition, it should be kept in mind that as the disease
progresses the individual in question may seroconvert to a
positive ANA, and that positive dsDNA tests can precede the
diagnosis of SLE by up to several years.(14) In fact, one
study evaluating the Farr assay found some individuals with
detectable anti-DNA titers up to 5 years before they were
diagnosed with SLE. (15)
Other diseases are known to produce positive anti-dsDNA titers.
Both rheumatoid arthritis and autoimmune hepatitis patients,
for example, have been known to have positive dsDNA test results,
but may not have a positive ANA test. (4,8) For this reason
it is important that all findings be interpreted in the context
of signs and symptoms. Because of our dependence on serological
tests to aid in the diagnosis of rheumatic diseases, it bears
repeating that positive tests without support of clinical
symptoms, patient history, and physical findings are of questionable
value.
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References
1. Informal survey of Immuno Concepts Technical Support customer
questions.
2. Manoussakis MN, Garalea KL, Tzioufas AG, Moutsopoulos HM.
Testing for antibodies to ENA and to dsDNA is not indicated
in FANA-negative sera. Clin Rheumatol 1988 Dec; 7(4):465-9.
3. Keren, DF. CAP today Q &A July 1998.
4. Werle E, Blazek M, Fiehn W. The clinical significance of
measuring different anti-dsDNA antibodies by using the Farr
assay, an enzyme immunoassay and a Crithidia luciliae
immunofluorescence test. Lupus 1992 Dec; 1(6):369-77.
5. Wong KH, Lawton JW, Cheng SK, Lee SS, Lau CS. Measurement
of anti-dsDNA: a comparative study of two ELISA and the Crithidia
assay. Pathology 1998 Feb; 30 (1):57-61.
6. Wigand R, Gottschalk R, Falkenbach A, Matthias T, Kaltwasser
JP, Hoelzer D. [Detection of dsDNA antibodies in diagnosis
of systemic lupus erythematosus—comparative studies of
diagnostic effectiveness of 3 ELISA methods with different
antigens and a Crithidia luciliae immunofluorescence
test]. Z Rheuatol 1997 Mar-Apr; 56(2):53-62.
7. Smeenk, R. Measurement of antibodies to DNA. In Van Venrooij
WJ, Maini RN, eds. Manual of Biological Markers of Disease.
London Kluwer Academic Publishers. 1993: A8/1-12.
8. Egner, W. the Use of Laboratory tests in the diagnosis
of SLE. J Clin Pathol 2000; 53:424-432.
9. Kumar V, Krasny S, Beutner EH. Specificity of the Crithdia
luciliae method for detecting anti-native DNA antibodies.
Effects of absorption for lipoproteins. Immunol Invest. 1985;
14: 199-210.
10. Deng JS, Rubin RL, Lipscomb MF, Sontheimer RD, Gilliam
JN. Reappraisal of the specificty of the Crithidia luciliae
assay for nDNA antibodies: evidence for histone antibody kinetoplast
binding. Am J Clin Pathol. 1984; 82:448-452
11. Hykema MN, van Bruggen MC, ten Hove T, de Jong J, Swaak
AJ, Berden JH, Smeenk RJ. Histone-containing immune complexes
are to a large extent responsible for anti-dsDNA reactivity
in the Farr assay of active SLE patients. J Autoimmun 2000
Mar; 14 (2): 159-68.
12. Halbert SP, Karsh J, Anken M. studies on autoantibodies
to deoxyribonucleic acid and deoxyribonucleoprotein with enzyme-immunoassay
(ELISA). J Lab clin Med 1981 Jan; 97(1): 97-111.
13. www.Lupus.org
14. Smeek RJ, van den brink HG, brinkman K, termaat RM, berden
JH, Swaak AJ. Anti-dsDNA: choice of assay in relation to clinical
value. Rheumatol Int 1991; 11(3):101-7.
15. Swaak T, Smeenk R. Detection of anti-dsDNA as a diagnostic
tool: a prospective study in 441 non-systemic lupus erythematosus
patients with anti-dsDNA antibody (anti-dsDNA). Ann Rheum
Dis 1985 Apr; 44(4): 245-51.
Will
Immuno Concepts’ slides work with other manufacturer’s
mounting medium?
Occasionally problems will arise from interactions
between another manufacturers’ reagents and our test
systems. Although many vendors sell reagents that appear compatible
with our slides, controls, and conjugates, we can offer no
guarantees that these alternative products will perform well.
Over the years numerous calls have been logged into Immuno
Concepts’ Technical Support regarding artifactual nucleolar
patterns, poor or variable staining intensity, film, non-specific
high background staining, and mechanical damage. In our experience,
many of these problems can be traced back to the use of other
manufacturer’s reagents—with mounting medium being
particularly problematic.
We have found three common problems associated with other
vendor’s mounting media: autofluorescence, poor or variable
staining intensity, and mechanical damage. Autofluorescence
is nonspecific fluorescence given off by the mounting medium
itself and generally appears as a film over the entire slide.
Poor or variable staining can be related to the pH of the
substituted reagent. Changes in acidity or alkalinity of the
mounting medium can greatly affect the appearance, preservation,
and intensity of fluorescence. Mechanical damage is often
associated with mounting media of high viscosity. An overly
viscous mounting media can tear the cells of the substrate
as the coverslip is moved. For these reasons our mounting
medium is standardized and adjusted for optimal results with
our test systems.
The problems mentioned above are unlikely to occur every time
an alternative reagent is substituted for its IC counterpart;
however Immuno Concepts’ slides perform optimally when
used with Immuno Concepts’ reagents. Technical Support
will do their best to help customers troubleshoot any problems
that occur, but we can only guarantee the performance of IC
kits in their entirety. As we are unable to predict the interactions
of other manufacturers’ products with our test systems
we may have difficulties accounting for abnormalities encountered
with substituted reagents.
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Can
Plasma, Pleural, Synovial, Cerebrospinal, or Pericardial Fluid
be used on Immuno Concepts’ ANA Test Systems?
Notice: The following information is not to
be construed as an endorsement of the use of specimens other
than the manufacturer’s recommended sample.
As written in our package insert the preferred specimen is
serum. We understand that upon occasion serum is not available
and the lab is faced with running an alternative sample such
as plasma, synovial, cerebrospinal, pleural, or pericardial
fluids. In the past several years Immuno Concepts Technical
Support has received an increasing number of calls requesting
information about the use of alternative samples in testing
for antinuclear antibodies (ANA) on our test systems.
We realize the difficulty involved in getting another draw
from a patient, and for that reason the final decision on
running a particular sample is left to the individual laboratory.
However, there are several key points that need to be considered.
Immuno Concepts ANA test systems were validated for use with
serum as the preferred specimen. Furthermore, the significance
of ANA patterns has been established only in sera. There is
some speculation that when inflammation occurs, autoantibodies
seep into other bodily fluids such as synovial, cerebrospinal,
pleural, or pericardial fluids. But, the point that remains
to be elucidated is the level at which they become clinically
significant.
The “normal” (low level clinically insignificant)
concentration range of autoantibodies in these samples has
yet to be established. Therefore, there is insufficient evidence
for Immuno Concepts to suggest a dilution or titer range for
laboratories to use. We suspect that the concentration of
ANAs present in the alternate specimens mentioned above will
differ from those typically found in sera. For this reason,
the recommended serum screening dilution of 1:40 may fail
to detect any ANAs in these fluids.
Disclaimer: Deviations from the manufacturer’s recommendations
outlined in the package insert, without prior procedure validation
in accordance with laboratory and regulatory body guidelines,
negates the results of the test. Immuno Concepts cannot support
any pattern interpretation or result obtained when using any
other fluid than the recommended serum. Should you decide
to use an alternate sample (of plasma, pleural, synovial,
cerebrospinal, or pericardial fluid) your report should contain
a disclaimer stating that this test was not run using the
manufacturer’s preferred specimen.
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Other Literature Available:
1. Wang Dy, Yang PC, Yu WL, Shiah DC, Kuo HW, Hsu NY. Comparision
of different diagnostic methods for lupus pleuritis and pericarditis:
a prospective three-year study. J Formos Med Assoc 2000 May;
99 (5):375-80.
2. Wang Dy, Yang PC, Yu WL, Kuo HW, Hsu NY. Serial antinuclear
antibodies titre in pleural and pericardial fluid. Euro Respir
J 2000 Jun; 15 (6):1106-10.
Khare V, Baethge B, Lang S, Wolf RE, Campbell GD. Antinuclear
Antibodies in Pleural Fluid. Chest 1994 106(3): 866-871.
3. Hazouard E, Legras A, Diot E, Ferrandiere M, Corcia P,
Ginies G. [Cerebrospinal fluid complement and antinuclear
antibodies in lupus meningoencephalitis]. Rev Neurol (Paris)
1998 Jul;154 (6-7):549-50.
4. Pollard KM, Furphy LJ, Webb J. Anti-Sm and anti-DNA antibodies
in paired serum and synovial fluid samples from patients with
SLE. Rheumatol Int 1988; 8(5):197-204
Will Immuno Concepts slides
work with other manufacturer’s PBS?
Occasionally problems will arise from interactions
between another manufacturers’ reagents and our test
systems. Although many vendors sell reagents that appear compatible
with our slides, controls, and conjugates, we can offer no
guarantees that these alternative products will perform well.
Over the years numerous calls have been logged into Immuno
Concepts’ Technical Support regarding artifactual nucleolar
patterns, poor or variable staining intensity, film, non-specific
high background staining, and mechanical damage. In our experience,
many of these problems can be traced back to the use of other
manufacturer’s reagents—with PBS being particularly
problematic.
The use of alternate PBS can result in an artifactual nucleolar
pattern, a generalized weak staining of the substrate, or
high-background staining. Most of these “problems”
can be linked to altered pH and ionic strength in other vendors’
PBS. Dramatic shifts in pH and ionic strength can alter protein
conformation and binding characteristics of antibodies, both
of which can account for increased background, an overall
weak staining of the substrate, as well as artifactual patterns.
The problems mentioned above are unlikely to occur every time
an alternative reagent is substituted for its IC counterpart;
however Immuno Concepts’ slides perform optimally when
used with Immuno Concepts’ reagents. Technical Support
will do their best to help customers troubleshoot any problems
that occur, but we can only guarantee the performance of IC
kits in their entirety. As we are unable to predict the interactions
of other manufacturers’ products with our test systems
we may have difficulties accounting for abnormalities encountered
with substituted reagents.
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