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.
Anti-Phospholipid Antibody Syndrome (Lupus Anticoagulant)
Autoimmune Progesterone Dermatitis
Churg-Strauss (Allergic Granulomatosis)
Interstitial Granulomatous Dermatitis with Arthritis
Lupus Erythematosus (SLE, DLE, SCLE)
Mixed Connective Tissue Disease
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.
Five basic patterns are recognized in indirect immunofluorescence:
Pattern Ab Directed Against Associations Homogeneous or diffuse nuclear staining Chromatin
Occasionally DS DNA
Drug induced SLE
Rim or peripheral staining DS DNA SLE Fine Speckled nuclear pattern Non-DNA nuclear constituents (Extractable nuclear antigens-ENA):
SCLE Coarse Speckled nuclear pattern Anti-centromere Scleroderma Nucleolar Nucleolar RNA Systemic sclerosis
Antigen Antibody System Disease % Positive Native DNA DS DNA SLE 40-60% Histones Antihistone Drug induced LE >95% Core proteins of small nuclear ribonucleoprotein particles (Smith antigen) Anti-Sm SLE 20-30% Ribonucleoprotein (U1RNP) Nuclear RNP Weak
SLE in 30-40% RNP SS-A (Ro)SS-B (La) Sjogren Syndrome 70-90%
Ro/SSA antigen is a 60 kDa protein attached to small RNAs to produce a 100 kDa complex with nuclear localization in normal adult skin
If insult such as UV, viral infection, or altered cytosolic calcium milieu occurs, Ro/SSA particles manifest upregulated or displaced expression from the nucleus to the cytosol and from the cytosol to the cell surface
This may explain the photosensitivity or photoreproduceability of skin lesions in anti-Ro/SSA positive LE patients and the worsening of disease symptoms in connective tissue disease patients experiencing viral illness
DNA topoisomerase I ScI-70 Systemic sclerosis 28-70% Centromeric proteins Anticentromere CREST 90% Histidyl-t-RNA synthetase Jo-1 Inflammatory myopathies 25%
Conditions Where ANA is intrinsic to the Diagnostic Criteria
Drug induced SLE 100% Autoimmune hepatic disease 100% Mixed connective tissue disease 100%
Diseases where ANA is useful for monitoring or prognosis
Juvenile chronic oligoarticular arthritis with uveitis 20-50% Raynaud phenomenon 20-60%
Diseases where ANA is not useful in diagnosis
Rheumatoid arthritis 30-50% Multiple sclerosis 25% Idiopathic thrombocytopenia purpura 10-30% Thyroid disease 30-50% Discoid lupus erythematosus 5-25% Infections Variable Malignancies Variable Silicone breast implant patients 15-25% Fibromyalgia 15-25% Relatives of patients with autoimmune disorders 5-25%
Titers > than or = to Percentage 1:40 20-30 1:80 10-12 1:160 5 1:320 3
Reference Methods Clinical Utility Interfering Diseases or Substances that Alter Levels Commonly Used Terms Internet Links
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.
CLINICAL UTILITY CHARACTERIZATION EARLY ONSET OF ANTI-dsDNA
Development of anti-dsDNA autoantibodies prior to clinical diagnosis of systemic lupus erythematosus.
Arbuckle MR, James JA, Kohlhase KF, Rubertone MV, Dennis GJ, Harley JB.
Arthritis and Immunology Program, Oklahoma Medical Research Foundation, Oklahoma City, OK 73104, USA.
Scand J Immunol 2001 Jul-Aug;54(1-2):211-9 Abstract quote
Anti-double stranded (dsDNA) antibodies are of considerable diagnostic value and are thought to be involved in the pathogenesis of systemic lupus erythematosus (SLE). Fluctuations in anti-dsDNA antibody levels are also used as markers for disease activity and exacerbations.
In this study we sought to evaluate the anti-dsDNA antibody level in serum samples collected before the onset of SLE diagnosis. A total of 130 SLE patients were identified with stored serum samples available prior to diagnosis within the US Department of Defense serum repository. All 633 sera available from these patients were screened for anti-dsDNA antibodies using an enzyme linked immunosorbant assay (ELISA). Within this cohort 55% of cases had detectable anti-dsDNA antibodies prior to SLE diagnosis. The onset of anti-dsDNA antibodies ranged from 9.3 years before to within the same month as diagnosis (with a mean onset 2.7 years before diagnosis).
In order to assess for fluctuations in anti-dsDNA levels relative to diagnosis, cases were selected with at least two positive samples, one within 6 months and a second greater than 6 months prior to diagnosis (n = 26). Seven of these cases also had samples available shortly after diagnosis (< or = 6 months) for comparison. Fifty-eight percent of the 26 cases developed a significant rise in anti-dsDNA antibody levels within 6 months of diagnosis. A significant decline in anti-dsDNA levels ensued after diagnosis (and following treatment with corticosteroids) in all seven cases with samples available. Patients with a significant rise in anti-dsDNA antibodies at diagnosis were more likely to have renal disease than those who did not (66.7% compared to 27.3%, chi2 =3.94, P<0.05).
These data suggest that anti-dsDNA antibodies are present in SLE patient sera much earlier than previously suspected. In addition, the data are consistent with increases in anti-dsDNA levels contributing to the onset of clinical illness in some patients with SLE.
INTERFERING DISEASES OR SUBSTANCES THAT ALTER LEVELS CHARACTERIZATION GENERAL
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.
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.
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.
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.
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.
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.
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.
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.
LOW DENSITY LIPOPROTEINS
Specificity of the Crithidia luciliae method for detecting anti-DNA antibodies. Effect of absorption for lipoproteins.
Kumar V, Krasny S, Beutner EH.
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.
MALARIA, ACUTE INFECTION
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.
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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HLA-Human Leukocyte Antigen. These are a group of proteins present on the majority of cells. They interact with the body's immune system, the T lymphocytes and involved in the induction and regulation of the system. The antigens are involved in transplant rejection as well as processing and eventual elimination of foreign proteins. Many diseases, including autoimmune disorders, are associated with these disease states.
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