A Basic Guide to Autoimmune Testing: Part I ANA, ENA and ...

1 A Basic Guide to Autoimmune testing : part I ANA, ENA and dsDNA Antibodies Typical scenario: A 40 year old woman presents with tiredness. She requests Autoimmune tests just to make sure , as a friend was diagnosed with Lupus some years back and has been quite unwell. She has looked it up on the internet. Blood tests reveal a normal full blood count, normal ESR, and lowish ferritin. Her ANA is 1/160, speckled pattern. A Reminder: The Clinical Manifestations of Lupus Systemic Lupus Erythematosus (SLE, or Lupus) is a complex Haematological Anaemia, low platelets, neutropenia Autoimmune disease, which may Photosensitivity, rashes, alopecia, Raynauds, acrocyanosis, present with a variety of clinical Skin mouth ulcers symptoms and signs.

2 This disease Synovitis, tendonitis is associated with various positive Joints (90% have some degree of joint involvement). antibodies, some of which are specific to Lupus, some of which indicate Renal Active urinary sediment, HT. another Autoimmune disease, and some of which can occur in healthy Heart, Lungs Pleurisy most commonly individuals. Recurrent late miscarriage, IUGR, recurrent or unexpected Thrombosis thromboembolic disease Antinuclear Antibodies (ANA). An ANA is an antibody against Cerebral Seizures, strokes a nuclear component of the cell. Constitutional Weight loss, fevers, fatigue At Clinipath Pathology, the test is performed by immunofluorescence, and Further elucidation of the specificity is tested against each one.

3 Seven a titre is given, as well as the pattern of of the antibody is done by ENA and antibodies are routinely tested for the fluorescence. dsDNA testing . These are useful in by ELISA at Clinipath. A rough Guide The ANA may represent many confirming the significance of a positive to the disease associations of ENAs autoantibodies, so once an ANA is ANA and will help to lead to a diagnosis is provided in Table 1. For some found, often further testing needs to be of the type of Autoimmune disease. antibodies, further confirmatory testing done to elucidate the type of antibody.

4 Extractable Nuclear Antigens may be required, (for example, for Jo 1. (ENAs) antibodies), as false positives may occur Titre Detecting antibodies to ENAs with the screening ELISA. Interpretation The titre is determined by the lowest involves testing patient's serum for in the clinical context is important. dilution at which the fluorescence can antibodies against various specific It is less likely that a patient still be seen. Hence, the higher the components of the cell nucleus. will develop clinically significant denominator, the stronger the intensity of The nuclear antigens are extracted Autoimmune disease if the ENAs are the fluorescence.

5 A 1/40 titre, therefore, individually, and the patient's sera all negative. is less significant than a 1/2560 titre. In Perth some laboratories give ANA. results as SI Units. With this method, Table 1. Main conditions which may be diagnosed from ANA, ENA testing the higher the SI Unit, the higher the intensity of the ANA. Due to differences True Positive ANA False Positive ANA . in methodology, unfortunately, it is not Nuclear Healthy individuals, especially age >60. possible to compare a result by titre with Systemic Lupus Erythematosus Infections one by SI Unit.

6 Sjogrens Syndrome Hepatitis C. Whether measured by titre or SI Unit, the higher the intensity, the higher the Scleroderma EBV. likelihood of underlying disease. Mixed Connective Tissue Disease HIV. Patients with a low titre ANA are Drug Induced Lupus Bacterial endocarditis likely to be healthy. Cytoplasmic Autoimmune diathesis . Pattern Polymyositis Rheumatoid arthritis Many different patterns can be Primary Biliary Cirrhosis Juvenile Chronic Arthritis detected using immunofluorescence, Autoimmune Hepatitis Hashimotos depending upon the specificity of the Graves Disease underlying antibody that constitutes Pernicious anaemia the ANA.

7 An example of these patterns is illustrated in Figure 2 over the page. Page 1 of 3. A Basic Guide to Autoimmune testing : part I ANA, ENA and dsDNA Antibodies continued Double stranded DNA Figure 1. How to interpret an ANA result titre (dsDNA). Likelihood of disease . Antibodies against dsDNA are highly Low titre ANA may occur in healthy individuals specific for SLE and are rarely found High titre has a high likelihood of significant Autoimmune disease in other disorders. They are useful for confirming the diagnosis of SLE, and 100% SLE. for monitoring disease. They predict an increased risk of Lupus nephritis.

8 They are only positive in a proportion of patients with Lupus. (~ 70%). Likelihood Monitoring SLE. Patients with Lupus may present with a variety of clinical problems, and when monitoring these patients, Healthy their particular disease often leads to signature parameters to follow. 1/40 Titre 1/2560. This will vary depending on the patient ( a patient with predominant Please don't hesitate to ask the 3. The Management of patients with unexpected Clinical Immunologist if you are not sure autoantibody positivity Bagnasco et al, Lupus nephritis will have different Autoimmunity reviews 2007 347-353.)

9 Monitoring parameters to a patient with how to proceed with further testing 4. British Columbia Guidelines for ANA testing Autoimmune haemolytic anaemia). for a patient in whom you suspect for connective tissue disease, 2001, updated ANA and ENA antibodies are not Autoimmune disease. It can be a 2007 useful for monitoring, and rarely need to complex field! be repeated after diagnosis. Tests used Dr Tiffany Hughes References Immunologist in monitoring are listed in Table 2. 1. The use of laboratory tests in the diagnosis of SLE Egner, W, J Clin Pathol 2000; 53:424-432 T: 9476 5222.

10 Table 2. Which tests are useful 2. Serologic testing in Connective Tissue E: Diseases Habash-Bseiso et al, Clinical for monitoring? Medicine and Research August, 2005. ANA 8. dsDNA 4. ENA 8. C3, C4 4. Urinary protein 4. Creatinine 4. ESR 4. CRP 8. Figure 2. ANA Speckled Pattern Table 3. Main Disease Associations with dsDNA and ENAs Anti dsDNA Specific for SLE. Anti SSA / Ro SLE, Sjogrens Syndrome (The babies of pregnant women with anti SSA are at risk of neonatal heartblock). Anti SSB / La SLE, Sjogrens Syndrome Anti RNP SLE/Mixed Connective Tissue Disease Anti Jo 1* Polymyositis/dermatomyositis Anti Sm Specific for SLE.