Advanced Practice Rheum: Antinuclear Antibodies (ANA) Save

Other videos in the Advanced Practice Rheum Series:

• Evaluation of Rheumatic Complaints

TRANSCRIPT:

Welcome to Advanced Practice Rheum. Hi, I’m Jack Cush with RheumNow. In this review we're going to cover antinuclear antibodies and autoantibodies in general. 

Antinuclear antibodies or ANA are basically equated with the diagnosis of lupus, but would be a mistake. While everyone with lupus is going to be ANA positive (99.9%),  finding a positive ANA test does not mean that you have lupus and we'll talk about why. 

The ANA test is very sensitive but poorly specific. The converse should also be considered: could you have lupus and be ANA negative? ANA negative lupus was uncommonly reported back between 1970 and 1984, and was related to how the test was done. After 1982, cellular substrates (mouse cell) that had no SSA antigens, were replaced with current cellular substrates (HEP2 cells, that have SSA antigens) and ANA negative lupus is hardly longer possible, and it's almost extinct. Such cases are now very rare - about 0.1% of all the ANAs ever done, ANA negative lupus should not be considered.

The ANA is really a test that tells you you're maybe dealing with autoimmune diseases and can be used to confirm such suspicions. It's very, very sensitive. Upwards of either 20 to 30 million Americans have a positive ANA in any titer, but less than 300,000 Americans have lupus. That means that only one in a hundred ANAs will be due to lupus. But there may be other diagnoses that may also cause ANA positivity. 

How is an ANA done? It used to be done by immunofluorescence testing, on a slide with cells exposed to patient's serum that has autoantibodies in it, maybe, and the antibodies would bind to nuclear antigens on the cells and then they would put on a secondary fluorochrome that would bind to the antibodies and they would read it under fluorescence.

Current faster methods are done by ELISA assays, which makes the test a lot more sensitive, meaning it's a lot more positive.  Cutoffs for what's positive is defined locally, testing a normal population and choosing a cutoff of roughly 5-6%. When you apply that cutoff to normal healthy people, 5 or 6% are going to have an ANA with basically zero risk of having lupus. However, if you apply the same cutoffs to an elderly population over 65, or a hospitalized sick population, it's going to be a threefold higher rate (~15%) of positivity. And that doesn't necessarily mean that they're going to have lupus. ANA positivity nonspecifically indicates immunologic activation. It is a very ineffective screen for lupus, especially in individuals with arthralgia or without lupus criteria.  Basically, you need a to have good reasons to consider a lupus diagnosis, then the test becomes a confirmatory, not screening, test. 

The ANA+  result may be found in other conditions; most commonly, no condition at all. Second, most common in my experience is autoimmune thyroid disease, Grave’s disease, Hashimoto's autoimmune thyroiditis in general. Next, autoimmune disease in general can be considered - myositis, polymyositis, dermatomyositis, systemic sclerosis, antiphospholipid syndrome, autoimmune hemolytic anemia, etc. Women who are pregnant have a doubling or tripling of their ANA+ results, found in  9 to 18% of pregnant women are going to be ANA positive but not have lupus. ANA positive results are commonly found with chronic renal disease, especially chronic liver disease. Chronic liver disease is notorious for giving you not just ANAs, but lots of autoantibodies including double stranded DNA. It's associated with neoplasia. So the differential diagnosis is quite wide on any positive ANA. 

We do know that if the ANA is used in general population as a screening test - it is poorly useful. It has really little predictive value. The significance of the test is very dependent on a few things, especially number one - the history.

The ANA is not significant if they don't have a history that doesn't suggest a diagnosis of lupus or another autoimmune disease, liver disease, lung disease, etc. The significance also rises with titer; titers of 1:40, 1:80 or 1:160 are weak, hardly enough to  care about - truly nonspecific. Titers greater than 1: 1280 up to 1:10,240, you should consider more seriously, but there is no high titer that is diagnostic.  After titer, you can consider the “pattern” of staining (see below). 

A  1972 Annals of Internal Medicine study showed if you had a several lupus criteria, the odds of getting lupus only went up until you had three or four criteria or more.  However, if you add an ANA to that equation, same thing; you have to have at least three criteria to make an ANA provide added diagnostic value in making the diagnosis of lupus.

So the indications, we said the causes: age is a cause (threefold increased risk); drugs are a cause, and there's drugs that just cause ANA positivity and drugs that also cause drug induced lupus. They are the same drugs that cause ANA positivity are more frequent than drugs that cause drug induced lupus. That's only a subset. It used to be drugs like procadimine and hydralazine. In more recent years, thorazine, lithium, quinidine, tetracycline and TNF inhibitors are notorious for causing positive ANAs. 

The next most common reason is no reason at all: if the ANA’s positive and without supportive lupus criteria, you can just follow the patient for future symptoms and not repeat the test unless the clinical scenario changes. When the history supports a lupus diagnosis, then an ANA test, in  high titers is useful and may support your testing for other autoantibodies beyond the ANA.. 

So what are the indications for ordering ANA? There's only one: a clear suspicion of lupus based on criteria and clinical presentation. In this instance, you would use the ANA to confirm your suspected diagnosis.

ofAfter that, you could order an ANA to evaluate the possibility of another autoimmune disease like scleroderma, myositis, or Sjogren's. In systemic sclerosis, 60-70% will be ANA positive. In myositis, about 50% are ANA positive. In Sjogren's, it's up to 80%. And, 40% of rheumatoid arthritis patients have ANA positivity as well. 

Arthralgia (or suspected arthritis) is not a reason to order an ANA. You'll have to spend a lot of time explaining why the ANA is positive in someone who just has arthralgia. Similarly, family history is not a reason, and a primary care doctors recommendation for ANA testing is also not a valid reason. 

While the sensitivity of an ANA is 98%, the specificity is only about 57%. This compares poorly to other more specific autoantibodies for lupus like double stranded DNA, which is found in 50 to 60% of SLE patients, but has a specificity of 97%. Also highly specific are  anti-SM antibodies, 97% specific, but only seen about 30% of lupus patients. RNP antibodies are not sensitive nor specific for lupus. 

Thus, you need to think about sensitivity and specificity when ordering these tests. Like the CCP antibody, a more specific test for rheumatoid arthritis (compared to the rheumatoid factor), double stranded DNA and anti Sm antibodies are much more specific tests for lupus compared to the ANA alone. 

A 2025 Journal of Rheumatology article looked at multiplex, ANA assays done on 2,500 individuals in one hospital system. These were ELISA tests primarily ordered by primary care doctors and next by other medical subspecialties for hundreds of reasons.

The vast majority of these were ANA negative, and only 1.6% of the ANA negative group ended up having an autoimmune rheumatic disease - most often rheumatoid arthritis or spondyloarthritis. In the ANA positive group, less than 14% were diagnosed with a new rheumatic disease or autoimmune disease, most often Sjogren's syndrome, lupus or undifferentiated connective tissue disease. Bottom line is that 97% of ANAs ordered by the general prescribing practitioners do not lead to an autoimmune or rheumatic diagnosis. So again, be selective in what you order. 

It may be helpful to note not just the titer but also the pattern of staining reported when the ANA comes back as positive. Most ANAs come back with a speckled pattern but that's horribly nonspecific. The antigens recognized are many: including RNP, Sm, SSA, SSB, etc. RNP has been associated with MCTD; Sm very specific for lupus; SSA (or Ro) is associated with Sjogren's; SSB (or La) associated with Sjogren's, but also other conditions. Jo-1 antibodies are associated with, myositis. Finding a speckled is very nonspecific; and you can gain further insight by ordering an extractable nuclear antigen (ENA)  test or individual titers of RNP, Sm, SSA, SSB, Scl-70, PM-1, Jo-1, etc. Also, non-specific is the diffuse pattern of ANA staining Most specific, as we said, is double stranded DNA antibodies that may yield a a peripheral or rim pattern of ANA. We don't see much of that anymore. 

Three things I want you to remember about patterns is: 1) speckled pattern is most common; 2) peripheral (rim) pattern is specific and associated with dsDNA antibodies; and 3) nucleolar or centromere patterns are uncommon, but have scleroderma associations. Nucleolar patterns are found in 40% of patients with systemic sclerosis (diffuse scleroderma), due to antibodies against RNA polymerase-1 and a few other antigens. The centromere pattern is seen in CREST syndrome (limited scleroderma) found in 75% of  limited systemic sclerosis.

You can sometimes get cytoplasmic staining which is also nonspecific and that can be sometimes confusing. 

ANA associates with lupus.  But it can be found in 3 types of lupus skin disease: 

1. Acute lupus skin disease (malar rash, alopecia, bullous LE, photosensitivity, oral ulcers). These patients are always ANA positive, may or may not have double stranded DNA and anti-Sm antibodies, and are at risk for organ damage (renal, lung, joints, etc).
2. Chronic lupus skin disease - usually referred to as discoid lupus. Only 50% of them are going to be ANA positive. These patients have almost no risk for organ disease and do not “progress” to acute or systemic lupus.
3. Subacute lupus (SCLE) skin disease - often with ANA antibodies; some of these patients will have antibodies against SSA (or Ro) and a low risk of organ involvement. 

ANA Rules:  

1. Don't order an ANA unless lupus criteria are present.
2. Don't repeat the ANA over and over and over again. Changing titers (especially if low) have no correlation with disease activity or future risk.
3. Don't order sub serologies (dsDNA, Sm, etc) if the ANA is negative. Subserologies should only be ordered if the ANA is positive.
4. ANA negative lupus is essentially extinct.
5. ANA positivity is variable in other autoimmune diseases, other connective tissue diseases ranging from about 40% to 80% positive.
6. Do not refer patients to rheumatology for low titer ANA (1:40, 1:80, 1:160) without other supportive features that would suggest an autoimmune disease. Such titers are meaningless and seldom pathogenic.

The ANA may also be found in patients with the “anti-phospholipid syndrome”.  Patients with this syndrome may be ANA positive and can have one of three differents antiphospholipid antibody tests: 1) Biologic false positive RPR (test for syphilis), 2) anti-cardiolipin antibodies, or 3) the lupus anticoagulant. The lupus anticoagulant can be tested in many different ways, but is suggested by the prolonged PTT, dilute Russel viper venom test or platelet neutralization test.

Lupus and autoimmune (RA, myositis, scleroderma) patients may have ANA and antibodies against phospholipids, without having the “antiphospholipid syndrome” which id defined as having any of the antibody tests (above) against antiphospholipid and one of three major manifestations of the antiphospholipid syndrome: 1) recurrent thrombotic events or major thrombotic events; 1) spontaneous abortions and miscarriages; and, 3)refractory thrombocytopenia. There are other features that go along with this: migraine; Raynaud’s; Libman-Sacks or sterile endocarditis; mitral regurgitation; transverse myelitis; or neuropathies. 

APL antibodies are not usually pathogenic, and they can be found in 30% or more of lupus patients. APL Ab tests are pathogenic with those clinical features I just mentioned. Also the presence of another autoantibody - beta-2 glycoprotein antibodies, with an antiphospholipid antibody test, increases the risk of having the anti-phospholipid syndrome, a disorder that is not treated with anti-inflammatory or immunosuppressive meds, but instead is treated ith chronic anticoagulation. 

Lastly, I'm going to talk about anti neutrophil cytoplasmic antibodies or ANCA. ANCA, as you know, is a test that was associated with Wegener’s granulomatosis, which is now called granulomatosis with polyangiitis (GPA). ANCA+ is found in 50 to 90% of GPA patients. There are 2 types of ANCA,  C-ANCA and P-ANCA. C-ANCA refers to diffuse cytoplasmic staining of the neutrophil cytoplasm. The antibody directly associated with a C-ANCA finding is antibodies to proteinase 3 (PR-3 Abs). If you're considering a GPA diagnosis, you should order C-ANCA or PR-3 antibodies. These tests have a moderate sensitivity, but high specificity for GPA. 

P-ANCA antibodies refer to a peri-nuclear pattern of immunofluorescence in the neutrophil cytoplasm.  P-ANCA antibodies are less specific and can be found in other conditions, including microscopic polyangiitis; eosinophilic granulomatosis with polyangiitis (EGPA, previously called Churg Strauss syndrome); and some patients with lupus and lupus nephritis. P-ANCA results are usually due to anti-myeloperoxidase (MPO) antibodies. 

One last point on how to order tests, especially when looking for autoimmunity or inflammation. Below are the American College of Physician recommendations about the use of the ESR. published in 1986, in the Annals Internal Medicine, here are the ESR guidelines from the American College of Physicians: 

1. The sed rate should not be used to screen asymptomatic people
2. The sed rate should be used selectively and interpreted with caution. Extreme elevation of the sed rate seldom occurs in people who have no evidence of serious disease or inflammatory disease
3. If there's no immediate explanation for an increased sedimentation rate, you should repeat the test at a later interval (some months later), rather than undertake a long, expensive, and worrisome search for some occult illness
4. The sed rate is useful in the diagnosis in monitoring of patients with polymyalgia rheumatica and giant cell arthritis.
5. In diagnosing, treating and monitoring patients with rheumatoid arthritis, the SED rate could be used to resolve conflicting clinical evidence. (meaning if you are unclear if the disorder is inflammatory or non-inflammatory, the ESR or CRP may help clarify)
6. The sed rate may be helpful in monitoring and treating patients with Hodgkin's disease. 

Tune in for more of these Advance Practice Rheum Reviews.

Editor’s note: This transcript has been prepared from the original recording and may include transcription inaccuracies. Please rely on the video for the most complete and accurate information.