An autoimmune disease is perhaps one of the cruelest of all diseases.
The body's normal immune system loses its ability to recognize its own cells
and attacks itself. The result is a devastating disease process which
spares no organ. This group of diseases is sometimes referred to as
collagen vascular diseases because the soft tissue supporting tissues
and blood vessels are frequently attacked.
It should be noted that this list is not complete and indeed, there are many
diseases found in other body sites which are also autoimmune in nature.
A good example is pemphigus vulgaris, a devastating blistering disease of
the skin and mucous membranes. Vasculitis, or inflammation of the blood
vessels, is also frequently autoimmune. These latter diseases are found
in the skin rash and blood
vessel pages.
Anti-nuclear antibodies are directed against nuclear antigens. They are
one of the hallmarks of an autoimmune disease. They can be broadly grouped
into four categories.
1. Antibodies to DNA
2. Antibodies to histone
3. Antibodies to nonhistone proteins bound to RNA
4. Antibodies to nucleolar antigens
These patterns are generated by employing an enzyme-linked immunoabsorbent
assay (ELISA). The immunofluorescence ANA pattern is generated by the overlay
of patient serum on a Hep2 cell or mouse epithelial cell-bearing glass slide.
This pattern correlates with the antibody specificity and direct immunofluorescence
findings in the skin biopsy material.
| REFERENCE METHODS |
CHARACTERIZATION |
Variability between methods to determine ANA, anti-dsDNA and anti-ENA autoantibodies: a collaborative study with the biomedical industry.
Bizzaro N, Tozzoli R, Tonutti E, Piazza A, Manoni F, Ghirardello A, Bassetti D, Villalta D, Pradella M, Rizzotti P.
Laboratorio di Patologia Clinica, Ospedale Civile, S. Dona di Piave (VE), Italy. |
J Immunol Methods 1998 Oct 1;219(1-2):99-107 Abstract quote
This study was performed by the Italian Society of Laboratory Medicine (SIMeL) in order to establish the variability between the different analytical systems currently used in clinical laboratories for the detection of autoantibodies diagnostic of systemic autoimmune disease.
Sixteen industrial, and two university laboratories participated in this study which entailed the determination of anti-nuclear (ANA), anti-dsDNA and anti-ENA antibodies in 11 sera from patients with clinically diagnosed systemic rheumatic disease, using reagents produced by these companies and different methodologies (indirect immunofluorescence, immunoenzymatic assay, counterimmunolectrophoresis, immuno and western blotting). We found 93.5% agreement between the methods used for the detection of ANA, 85.2% for anti-dsDNA antibodies, and 86.9% for anti-ENA antibodies. Among the anti-ENA antibodies, regardless of the method used, detection percentages were excellent for anti-RNP and anti-SSB/La (100%), good for anti-SSA/Ro (93%), but unacceptable for the anti-Jo-1 (67%), anti-Scl70 and anti-Sm (47%) antibodies.
This further stresses the need for rigorous standardisation of commercial reagents and analytical procedures, as well as the introduction of external quality assessment (EQA) programs, and a complete definition of operative protocols adjusted to the sensitivity and specificity of the various methods. |
Comparison of an enzyme immunoassay to an indirect fluorescent immunoassay for the detection of antinuclear antibodies.
Reisner BS, DiBlasi J, Goel N.
University of Texas Medical Branch, Galveston, USA. |
Am J Clin Pathol 1999 Apr;111(4):503-6 Abstract quote
The standard method for detecting antinuclear antibodies (ANAs) is by immunofluorescence assay (IFA), a method that is labor intensive and subjective. In an attempt to overcome these limitations, several commercial enzyme immunoassays (EIAs) have been developed.
We report the results of our evaluation of the ANA Microplate EIA (Sanofi Diagnostics Pasteur, Chaska, MN). For the evaluation, 808 serum samples were tested by EIA and IFA; 52 specimens were positive by both assays, 561 were negative by both assays, 91 were positive by EIA only, and 3 were positive by IFA only. Borderline results (not positive or negative) were obtained for 101 specimens, which were excluded when calculating the sensitivity, specificity, and positive and negative predictive values of this assay, which were 94.6%, 86.0%, 36.4%, and 99.5%, respectively.
Because of its high negative predictive value, this assay can be used reliably to detect ANA-negative samples; however, the low positive predictive value indicates that EIA-positive specimens should be retested by an IFA to determine the final result. |
Guidelines for the Laboratory Use of Autoantibody Tests in the Diagnosis and Monitoring of Autoimmune Rheumatic Diseases
Renato Tozzoli, MD
Nicola Bizzaro, MD
Elio Tonutti, MD
Danilo Villalta, MD
Danila Bassetti, MD
Fabio Manoni, MD
Anna Piazza, MD
Marco Pradella, MD
and Paolo Rizzotti, MD |
Am J Clin Pathol 2002;117:316-324 Abstract quote
The Italian Society of Laboratory Medicine Study Group on the Diagnosis of Autoimmune Diseases has generated a series of guidelines for the laboratory diagnosis and monitoring of systemic autoimmune rheumatic diseases intended for the use of clinical pathologists and laboratory physicians.
These guidelines are based on a systematic review of published works and expert panel discussion and consist of 13 recommendations for antinuclear antibodies, anti–double-stranded native DNA, and antinuclear specific antibodies.
To improve analytic performances and help select the most appropriate test for specific autoantibodies, as well as provide education and guidance in the use of these tests, special emphasis is placed on laboratory methods. |
| INTERFERING DISEASES OR SUBSTANCES THAT ALTER LEVELS |
CHARACTERIZATION |
| GENERAL |
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Complete saturation of protamine sulphate by dsDNA is necessary
in order to obtain a highly sensitive and specific anti-dsDNA ELISA.
Rupin A, de Jong W, Degenne D, Bardos P.
Laboratory of Immunology, Faculte de Medecine, Tours, France.
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J Immunol Methods 1993 Apr 2;160(2):245-52 Abstract quote
A protamine sulphate (PS) pretreated solid phase coated with different
amounts of dsDNA has been used to develop a sensitive, specific and
reproducible anti-dsDNA ELISA.
Using low concentrations of a dsDNA coat 50% of SLE sera were found
to be positive and false positive reactivity due to anti-PS reactivity
was found in 3/40 patients with other auto-immune diseases (OAID). In
contrast, when PS was saturated with higher concentrations of dsDNA
80% of SLE sera were detected, the reproducibility of the results was
better and anti-PS reactivity of OAID patients with an anti-PS reactivity
disappeared. The sera of three other OAID patients contained low avidity
anti-dsDNA, measured after a salt elution step in the ELISA procedure,
and 2/60 patients with non-auto-immune disease exhibited a false positive
anti-dsDNA reactivity since they reacted with the solid phase even in
the absence of PS and dsDNA.
Thus an ELISA procedure using a PS pretreated solid phase permits the
sensitive, specific and reproducible measurement of anti-dsDNA antibodies
only if a high concentration of dsDNA is coated on the PS and appropriate
controls are performed.
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Differences in clinical sensitivity of ELISA tests for autoantibodies
with human and bovine extractable nuclear antigens.
Kapogiannis B, Gussin HA, Teodorescu MR, Teodorescu M.
University of Illinois College of Medicine, Department of Pediatrics,
Chicago 60612, USA.
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Lupus 2000;9(5):343-52 Abstract quote
Bovine antigens are routinely used in indirect ELISA tests to detect
autoantibodies against extractable nuclear antigens (ENA).
Here we investigate the difference in clinical sensitivity between
ELISA tests prepared with native human and bovine antigens. SSA and
SSB were obtained from spleen and nRNP/Sm complex from thymus. Each
antigen was extracted with the same immunoaffinity column. ELISA tests
with human and bovine antigens were set up under the same conditions
of clinical specificity established on 50 blood bank donors.
Of 109 random SLE and Sjogren's syndrome sera 49% and 35% were positive,
respectively, for human and bovine SSA, 26% and 16% for SSB. Of 98 random
SLE sera 52% and 41% were positive for human and bovine nRNP/Sm, respectively.
A few specimens reacted only with bovine antigens, probably false positive
reactions. The relative clinical sensitivity for all specimens identified
as positive by human and/or bovine antigens was significantly higher
with human than with bovine SSA, (93% vs 67%; P<0.001, chi2), SSB (93%
vs 50%; P<0.001), and for nRNP/Sm (96% vs 75%; P<0.01). However, for
values that exceeded 2.5-4 times the upper normal limit, the levels
were similar for human and bovine antigens.
We concluded that native human antigens offer clinical sensitivity
superior to native bovine antigens for the measurement of anti-ENA antibodies
by ELISA.
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| AGE |
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Anti-double-stranded DNA antibodies in the healthy elderly: prevalence
and characteristics.
Ruffatti A, Calligaro A, Del Ross T, Bertoli MT, Doria A, Rossi
L, Todesco S.
Division of Rheumatology, University of Padova, Italy.
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J Clin Immunol 1990 Nov;10(6):300-3 Abstract quote
Using Crithidia luciliae fluorescent assay a significant prevalence
(7.6%; P less than 0.006) of anti-double-stranded DNA antibodies was
found in a healthy old population. A negative enzyme-linked immunosorbent
assay for anti-total histone antibodies excluded a false-positive reaction.
Anti-double-stranded DNA antibodies in the aged differed from those
found in patients with systemic lupus erythematosus and were characterized
by a low titer (95.6% of cases), belonging to the IgA class alone (95.6%),
no complement-fixing ability (100%), and negativity to Farr assay (100%).
It is concluded that, in elderly subjects without signs and symptoms
of disease, including systemic lupus erythematosus, such a peculiar
anti-double-stranded DNA antibody may be detected.
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| HIV INFECTION |
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Anti-nuclear, anti-neutrophil cytoplasmic and anti-glomerular basement
membrane antibodies in HIV-infected individuals.
Savige JA, Chang L, Horn S, Crowe SM.
University Department of Medicine, Austin Hospital, Heidelberg,
Victoria, Australia.
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Autoimmunity 1994;18(3):205-11 Abstract quote
Many autoantibodies have been described in HIV-infected individuals.
We have examined the incidence, associations and prognostic significance
of anti-nuclear antibodies (ANA), anti-neutrophil cytoplasmic antibodies
(ANCA) and anti-glomerular basement membrane (GBM) antibodies in individuals
with HIV infections.
One hundred and five patients, with asymptomatic infections (n = 37),
AIDS-related complex (n = 32) or AIDS (n = 36) were studied.
Plasma from 24 of these (23%) were positive for ANA: most demonstrated
speckled fluorescence (n = 21) and were of low titre (1+ in 18). ANCA
were demonstrated by IIF in 18 individuals (17%) and all fluorescent
patterns were seen; 6 of these plasma were also positive in the ELISAs
for antibodies to proteinase 3, myeloperoxidase or elastase. Thirteen
plasma were positive for ANCA in the neutrophil cytoplasm ELISA; 10
of these were also positive in the specific ELISAs. A total of 30 plasma
bound to proteinase 3, myeloperoxidase or elastase in specific ELISAs,
in 6 cases with 2 specificities. Finally, 18 plasma (17%) contained
anti-GBM antibodies by ELISA, but none of 4 plasma tested in inhibition
assays was specific.
ANA, ANCA and anti-GBM antibodies were not uncommon in HIV-infected
individuals but the presence of these antibodies was not associated
with the clinical manifestations of the corresponding autoimmune diseases.
In addition, there was no correlation between the demonstration of these
antibodies and the immunological status of the individual (apart from
a correlation between CD4 counts less than 400/microliters with anti-GBM
antibodies), the presence of an opportunistic infection, the development
of malignancy or reduced survival. Some of these antibodies may arise
from polyclonal activation, or be due to "sticky" serum since we have
shown that the presence of anti-GBM antibodies correlated with the demonstration
of ANCA by ELISA. These antibodies are not more common in hypergammaglobulinemic
plasma but some may be due to heat-treatment of the plasma.
The clinician caring for HIV-infected individuals needs to be aware
of these "false-positive" antibody results.
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| LOW DENSITY LIPOPROTEINS |
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Specificity of the Crithidia luciliae method for detecting anti-DNA
antibodies. Effect of absorption for lipoproteins.
Kumar V, Krasny S, Beutner EH.
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Immunol Invest 1985 Jun;14(3):199-210 Abstract quote
Using the immunofluorescent (IF) assay with Crithidia luciliae smears,
anti-native (n) DNA antibodies were detected in the sera of 12 of 20
systemic lupus erythematosus (SLE) patients, in 1 of 6 mixed connective
tissue disease cases, in 2 of 38 patients with systemic sclerosis but
in none of the sera from 96 normal subjects. All anti-nDNA antibodies
were associated with antinuclear antibodies (ANA). However, occasionally
sera were encountered in routine screening which appear to be positive
for anti-DNA antibodies but negative for ANA.
Studies of such sera indicate that this is a nonspecific reaction which
can be abolished by treating sera with dextran sulfate or heparin. Treatment
of SLE sera with these agents had no effect on their anti-nDNA antibody
activity. Absorption of sera with Aerosil eliminated the false positive
reactions with C. luciliae; however, this treatment also removed immunoglobulins,
ANA and anti-nDNA antibodies.
Evidence is reviewed which points to a role of complexes of low density
lipoprotein and IgG in the nonspecific binding reactions with C. luciliae
which is seen as false positive reactions for anti-nDNA antibodies.
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| MALARIA, ACUTE INFECTION |
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Autoantibodies, immunoglobulins, complement and circulating immune
complexes in acute malaria.
Jhaveri KN, Ghosh K, Mohanty D, Parmar BD, Surati RR, Camoens HM,
Joshi SH, Iyer YS, Desai A, Badakere SS.
Government Medical College and New Civil Hospital, Surat, Gujarat,
India.
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Natl Med J India 1997 Jan-Feb;10(1):5-7 Abstract quote
BACKGROUND: Malaria caused by Plasmodium vivax and Plasmodium falciparum
is common in the Indian subcontinent. Studies conducted elsewhere have
suggested that malarial infection causes intense immunostimulation.
We screened patients with malarial infection for autoantibodies and
measured the immunoglobulin, circulating immune complex and complement
levels to determine the extent of immunological alterations in these
patients.
METHODS: One hundred adults with acute malarial infection confirmed
by examination of the peripheral blood smear and 25 age- and sex-matched
controls were studied. An autoantibody screen and serum immunoglobulin
complement (C3 and C4) and circulating immune complex levels were measured
at the time of admission and 4 weeks after they became afebrile. A direct
Coomb's test was also done.
RESULTS: Anti-ssDNA, anti-dsDNA and rheumatoid factor were positive
at the time of admission in 51, 30 and 38 patients respectively. None
of the controls were positive for these autoantibodies except for one
who was positive for rheumatoid factor. The IgM, IgG and IgA levels
were raised in 16, 25 and 36 patients respectively. Circulating immune
complex levels were raised in 32 patients and complement C3 and C4 were
low in 8 and 31 patients. Follow up studies at 4 weeks in 19 patients
showed that the autoantibodies were negative. However, the immunoglobulin,
C4 and circulating immune complex levels remained elevated. Six per
cent of patients had a positive direct Coomb's test with reticulocytosis
at the time of presentation.
CONCLUSION: Acute malarial infection can cause false-positive results
for anti-ssDNA, anti-dsDNA and rheumatoid factor and may also cause
a rise in the serum immunoglobulin, complement and circulating immune
complex levels.
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