ANA / ENA

  1. Do histone antibodies occur without other ANAs?

    "Histone antibodies are amongst the most abundant in health and disease. They tend to arise with ageing and chronic infection, and as a response to therapy with various drugs. They may occur in rheumatoid arthritis and juvenile chronic arthritis. Titres are almost invariably high in drug induced lupus, and histone antibodies are particularly common in systemic lupus erythematosus and autoimmune chronic active (lupoid) hepatitis; they are probably responsible for the LE cell phenomenon." (Citation from Bernstein R, Autoantibody Manual C2.2, 1996).

    Nevertheless, in our application lab in most cases histone antibodies are occurring together with anti-dsDNA antibodies (being positive in Varelisa, thus maybe low avidity dsDNA antibodies). Only in very rare cases we are finding a homogeneous pattern (HEp-2) in infections or healthy blood donors.

  2. Why are EliA dsDNA and Varelisa dsDNA Antibodies only against IgG and not IgG and IgM?

    IgM for dsDNA are, similar to IgG, a heterogeneous group of antibodies, some with high avidity, some with low avidity. Farr RIA is a highly specific assay for SLE which measures only IgG and IgM antibodies with high avidity. Low avidity antibodies are not found with this assay. ELISAs in general find antibodies of both, low and high avidity, which leads to a lower specificity but higher sensitivity of these assays.
    Anti-dsDNA IgM antibodies have a lower specificity than IgG, particularly when they are of low avidity. Determined by ELISA, they have been demonstrated in a variety of diseases like Sjögren's syndrome, scleroderma, MCTD and chronic active hepatitis.

    Specificity is a big aim in autoimmune diagnostics, particularly - to ensure the highest specificity possible, we do not measure IgM.

    Please find some articles to this topic here:

  3. Why does the EliA ANA positive control give a negative result?

    You should not dilute the ANA Control

    Please enter a request (sample id),
    then select a method (EL-G),
    then input a testname (sy),
    then press "shift enter" instead of "enter",
    then you can select "prediluted" instead of "instrument dilution" (dilution factor 100 is OK for sy)

  4. Do we have sensitivity and specificity data for all EliA analytes?

    Sensitivity and specificity data for CCP, Celikey and ANCA on EliA are available. Sometimes taken from external studies, but e.g. for ANCA we have done them internally before launch. 

    Some of the EliA analytes are markers of very rare diseases and are found only in a portion of patients. Obviously, for those markers it is not possible to define clinical sensitivity and specificity when developing such a test.
    For those analytes a technical sensitivity and specificity was evaluated by measuring samples with defined antibody specificity such as AMLI samples, CDC samples and WHO samples.
    Of course the data for technical sensitivity and specificity are not at all comparable to clinical sensitivity and specificity.

  5. Is EliA dsDNA suitable for the monitoring of SLE?

    dsDNA as monitoring marker is not mentioned as intended use in our DfUs. However, it has been shown in several studies, that EliA dsDNA reflects the disease activity and the involvement of the kidney. Therefore it can be used as monitoring marker.  In the following, the references are sorted by publishing data:

    Hernando M et al. Clinical Evaluation of a New Automated Anti-dsDNA Fluorescent Immunoassay.

     

    Varelisa

    EliA

    Farr

    CLIFT

    Sensitivity for SLE (%)

    44.7

    39.5

    31.6

    13.2

    Sens. for active SLE (%)

    70.8

    70.8

    66.7

    29.2

    Sens. for nephritic SLE (%)

    70.0

    55.0

    50.0

    25.0

    Specificity (%)

    82.5

    93.2

    96.1

    99.0

    • "In conclusion, the EliA™ dsDNA assay showed a very good specificity for SLE and a good sensitivity for active SLE. This assay can be used for diagnosing SLE and also for the follow-up of the disease activity."
    • "However, in clinical laboratories in which the Farr assay cannot be employed,the good sensitivity, high specificity, analytical accuracy, high degree of automation, and simplicity of execution of the EliA dsDNA test make it particularly appropriate for both the diagnosis and the monitoring of SLE patients."
    • "When compared with the ELISA, the newly developed FIA had better correlation with the serial changes of SLEDAI and C4, and was inversely correlated with the serum levels of C3. In addition, FIA had the advantage of better correlation with the occurrence of nephritis. It is worth developing this method for clinical evaluation of disease activity in SLE patients."
    • "In this retrospective study of a large cohort of patients, the new EliA dsDNA assay was demonstrated to be of slightly more diagnostic value for detecting lupus flares than the CLIFT. These data, together with the easier handling of the method, make EliA dsDNA a suitable tool for monitoring of SLE patients."
    • "CLIFT assay is likely to be accurate...; however, even though easy to perform, it is greatly operator-dependent and semiquantitative in antibody level measurement. Antibody quantitation by serum titration is considered inadequate in measuring changes in anti-dsDNA levels during disease course."
      "The ROC curves with our data showed that the EliA assay performs better than the Farrzyme dsDNA test."
    • "As regards SLE clinical monitoring, the assays gave meaningful results, generally concordant one to the other in realtion to global activity and organ specific involvement, mostly renal and haematologic, as expected. In addition, antibody levels correlated with global disease activity score measured by ECLAM score, in an independent manner."
  6. ENA vs ANA definition

    Definition ANA

    ANA (antinuclear ANTIBODIES) classically are detected by immunofluorescence. Thus they are named after their place where they bind the antigens - in the nucleus. It is a general name for all antibodies which react with nuclear antigens.
    But in the practical termination of doctors ANA also can bind antigens in the cytoplasm (like Jo-1 and Rib-P).

    Definition ENA

    ENA (extractable nuclear ANTIGENS) are a wide spectrum of nuclear antigens, which can be eluated easily by salt extraction. ENA are usually commercially available rabbit or calf thymus extract (CTE). They commonly has been used for Ouchterlony assays, but can be used also in other methods than immunodiffusion. ENA for ELISA or RIA are also commercially available, but their purity is not known. Usually, recombinant nuclear antigens are not called ENA.

    The diagnostically most important ENAs are the Sm antigens. Other ENAs are U1-snRNP, SS-A/Ro, SS-B/La, Jo-1, Scl-70, PM-1, Mi and Ku.

    DNA, centromere proteins and histones don't belong to the group of ENAs, but are nuclear antigens.

    ENA or ANA

    In summary the term ENA is used for a special group of nuclear antigens, defined by the method of purification. ANA can bind to ENA but also to other nuclear antigens.

    I think the term "ANA Screen" is more correct than "ENA Screen" (even if we used only ENA for coating), as we mostly use recombinant antigens, not extracted antigens from animal tissue. (Besides we screen for antibodies, not for antigens).

    More difficult is the situation in France or other countries were the term ANA is only used for ANA detected by immunofluorescence. The laboratory is obliged to use immunofluroescence if the physician asks for an ANA Screen. Only if ENA is asked for, the laboratory has the possibility to use ELISA.

  7. What is the clinical association of antibodies to the different U1 antigens?

    Anti-U1 RNP antibodies are found in about 30 to 40 % of patients with SLE. These antibodies may occur alone but are often present in conjunction with other specificities. Although these antibodies may occur alone in SLE, the converse finding, patients with anti-Sm who lack anti-RNP, is unusual.

    The major clinical association of anti-U1 RNP antibodies is with mixed connective tissue disease (MCTD) where they typically occur in high titers and are not associated with other specificities. Indeed, this illness is defined by the presence of these antibodies.

    Anti-U1 RNP antibodies may also occur in a small fraction of patients with Sjögren¿s syndrome, rheumatoid arthritis, scleroderma, and polymyositis.

    Longitudinal studies have indicated that anti-U1 RNP antibody titers vary over time, but it is uncertain whether these levels reflect underlying disease activity.

    Different antibody populations

    The three unique proteins of U1 snRNP, 70 kD, A, and C, do not share known cross-reactive epitopes, and are recognized by at least three separate antibody populations, which may occur together or singly in a given patient. In other words, all three antibodies contribute to the anti-U1 RNP response.

    When patient sera are grouped and examined irrespective of disease, anti-70kD, anti-A and anti-C antibodies appear to have similar prevalences.

    Anti-70 kD U1 RNP
    In general, anti-70 kD antibodies are found in around 12 % of patients with SLE.

    As few as 8% to 21%, up to 85% of preselected anti-U1 positive SLE patients will have anti-70 kD antibodies, depending on the sensitivity of the assay. Anti-70 kD occur in 75 to 95 % of patients with MCTD (who are, per definition, preselected as anti-U1 RNP positive).

    When patients with MCTD and SLE are grouped together, anti-70 kD antibodies appear to correlate with myositis, esophageal hypomotility, Raynaud¿s phenomenon, lack of nephritis, and the HLA-DR4 phenotype.

    Anti-A RNP
    Among patients selected because they have SLE, anti-A antibodies appear to be twice as common as anti-70 kD antibodies, appearing in approximately 23% of such patients overall, or in approximately 75% of preselected anti-U1 positive SLE patients.

    Presently, it is unclear, whether anti-A antibodies are associated with specific disease manifestations.

    Anti-C RNP
    Clinical associations of these antibodies have not been recognized as yet.

  8. What is the Wo80 standard and how can kits have different cut-offs although they are standardised against the same international standard?

    The Wo/80 Standard is the international standard of the WHO for measuring of anti-dsDNA antibodies. All anti-dsDNA kits which are calibrated against this standard, do use Internation Units (IU/ml) instead of arbitrary units (which is the common quantitative unit in autoantibody testing, because only in very few cases a standard is available).

    The Wo/80 is human serum with dsDNA antibodies. The standard has existed since 1985 and is marketed by the Central Laboratory of the Netherlands Red Cross Blood Transfusion Service (CBL) in Amsterdam.

    All kits which are calibrated against the Wo/80 Standard have to find it at a level of 100 IU/ml.
    Both Varelisa dsDNA Antibody test and EliA dsDNA are calibrated against the Wo/80 standard.

    However, both kits have different cut-offs: EliA dsDNA at 15 IU/ml and Varelisa dsDNA Antibodies at 55 IU/ml. This is due to the high heterogeneity of dsDNA antibodies: Some patients make low affinity antibodies that do not bind tightly, do not form very stable complexes or the complex can be separated by minor changes in temperature, ionic strength, pH etc.
    Some patients make low affinity antibodies that do not bind tightly AND high affinity antibodies that do bind tightly.

    What is the result? Every sample has the potential to behave differently from:

    • every other sample
    • samples at different times from a patient
    • any standard
    • any quality control material
    • any quality assurance material

    This results in a huge variability in results between

    • labs
    • methods
    • samples
    • concentrations

    Thus, although all tests are standardised against Wo80 and give international units, the result can vary widely between different patient samples. In quality assessment schemes it can be seen that one sample can produce very different results, depending on the method used.

  9. Is it often seen, that La antibodies are positive, but Ro abs are negative?

    Anti-SS-B/La antibodies are found almost always in combination with anti-SS-A/Ro antibodies and in 10 to 20 % of cases the anti-SS-A/Ro antibodies are found solely. (1) Neonatal lupus erythematosus (NLE) rarely occurs in the absence of Ro antibody; only four cases of La antibody positivity in the absence of Ro antibody have been reported. However, three of the four mothers were positive for both anti-Ro and anti-La antibodies. It is extremely rare to find La antibody in the absence of Ro antibody in both mother and infant. One case is described in the article of Neidenbach et al. (2) The significance of the finding of solely La antibody positivity is unknown, but it does support observations by Provost et al. that La antibody is significant in the pathogenesis of NLE. (2) Franceschini et al. describe an infant with cutaneous lupus lesions who, along with his mother, was positive for antibodies to La and negative for anti-Ro and anti-U1 RNP antibodies. (3) In 1989 Buyon et al. found, that antibodies to the 48-kD La antigen and its 43-kD degradation product were both found in the highest prevalence and were the antibody species most strongly associated with NLE. The individuals without detectable antibodies either to 52- or 60-kD Ro antigens each had very high levels of antibodies that reacted with the La polypeptides.

     

    References:
    (1) Humbel RL (1998) Autoantikörper und Autoimmunerkrankungen, Elsevier, Amsterdam

    (2) Neidenbach PJ, Sahn, EE (1993) La (SS-B)-positive neonatal lupus erythematosus: Report of a case with unusual features. J Am Acad Dermatol 29, 848-852

    (3) Franceschini F, Bertoli MT, Martinelli M et al. (1990) The neonatal lupus erythematosus associated with isolated La(SSB) antibodies. J Rheumatol 17, 415-417

    (4) Buyon et al. (1989) Acquired Congenital Heart Block. J Clin Invest 84, 627-634 

    References:
    (1) Humbel RL (1998) Autoantikörper und Autoimmunerkrankungen, Elsevier, Amsterdam

    (2) Neidenbach PJ, Sahn, EE (1993) La (SS-B)-positive neonatal lupus erythematosus: Report of a case with unusual features. J Am Acad Dermatol 29, 848-852

    (3) Franceschini F, Bertoli MT, Martinelli M et al. (1990) The neonatal lupus erythematosus associated with isolated La(SSB) antibodies. J Rheumatol 17, 415-417

    (4) Buyon et al. (1989) Acquired Congenital Heart Block. J Clin Invest 84, 627-634

  10. Why don't we put Histones on EliA?

    Reasons for discontinuing Histone Abs:

    1. The right antigen is not known
    2. Histone Abs are not specific for any disease
    3. Diagnosis of drug-induced lupus can be made without histone antibodies
    4. Positivity for AHA is often misinterpreted.

    Details to these points see below. Further arguments are:

    1. Patient-to-patient variability is very high
    2. Association with disease activity in lupus or RA is possible but not usable for monitoring
    3. No association with specific manifestations
    4. A negative result for IgG antihistone antibodies does not exclude a diagnosis of drug-induced lupus or SLE

    1. The right antigen is not known

    For drug induced lupus it is said, that antihistone antibodies (AHA) react with all histones, but have a predominant specificity to H1 and H2B. The classical ELISA for AHA is coated with a mixture of all histones. It is not known, whether those are coated as complex or as single proteins or if the proteins are in a natural or denatured conformation.

    Some authors claim that antibodies against the (H2A-H2B)-DNA complex are the "real" markers for drug-induced lupus (DIL). But what is the difference of this complex to the nucleosome? Nucleosomal antibodies are said to be highly specific for SLE, not for DIL. The dimer H2A-H2B seems to much less specific and sensitive for DIL. DNA lacks here - but DNA-antibodies are very specific markers for SLE and are an exclusion criterion for DIL.

    On the congress in Geneva in February, one lecturer spoke about H1 antibodies being very specific for SLE. But if they are the typical antigen for histone antibodies and these are typical for DIL ¿ than what is the truth?

    2. Histone antibodies are not specific for any disease

    Histone Abs belong to the most common ANA. In some connective tissue diseases they occur regularly and in a high amount:

    -  SLE                                         50-70%
    -  Active SLE                                80%
    -  Rheumatoid arthritis (RA)          15%
    -  Juv. chronic arthritis (JCA)          50-70%
    -  Sjögren`s Syndrome                  23%
    -  Polymyositis/Dermatomyositis    17%

    But they occur also often in Felty's syndrome, diffuse cutaneous scleroderma, systemic sclerosis, autoimmune disorders of the liver, some neurological diseases, infections and even in asymptomatic relatives of SLE-patients.

    And of course they are found in drug induced lupus, but dependent on which drug the lupus induced, the frequency of histone abs varies:

    -  Procainamide induced lupus         85-95%
    -  Quinidine induced lupus               50%
    -  Hydralazine induced lupus            approx 30%       

    3. Diagnosis of drug-induced lupus is more accurate without measuring histone antibodies

    The diagnosis of DIL should base on the following criteria:

    • Signs or symptoms of SLE in a patient on lupus-inducing drug therapy
    • No multisystem involvement or serious CNS or renal involvement
    • Improvement and resolution of symptoms within days to weeks of discontinuing drug therapy
    • No dsDNA antibodies (no Sm, RNP, Ro, La)
    • No hypercomplementemia

    4. Positivity for AHA is often misinterpreted

    AHA often are understood as markers for SLE and drug-induced lupus. But because histone Abs can occur in a wide variety of diseases and even in healthy individuals, the positive result is more misleading than helpful.

As in all diagnostic testing, the diagnosis is made by the physican based on both test results and the patient history.