Unfortunately, if the ANA test was run using a very common method called Multiplex testing, recent research indicates that there is an up to a 43% chance (depending on the particular Multiplex diagnostic system) that the negative ANA is incorrect (a false negative) for patients with scleroderma!
Diagnosis – Step 1: ANA Testing
(This next discussion is a bit technical but VERY important, so hang in there…).
For many years, ANA testing was done using a method called indirect immunofluorescence (commonly abbreviated as IFA or IIF). This has very high reliability and is the best way to test for the presence of anti-nuclear antibodies. However, it is a complex and time-consuming test that depends on highly trained laboratory personnel. Recently, many commercial laboratories and some larger hospital laboratories have switched their routine ANA and follow-up specific antibody testing to solid phase immunoassays (ELISA or EIA) or a related technique known as a Multiplex platform. These new techniques are less expensive than IFA. However, these new methods of testing can only detect a limited subset of the specific antibodies that are targeted by the tests (typically 8-13) in contrast to IFA that can detect up to 150 different possible antibodies. As a result, these alternate testing methods are more likely to miss relevant autoantibodies yielding a false negative ANA result, as indicated above. This can have a major impact on scleroderma diagnosis. If the results of an initial ANA screening come back negative to the doctor who ordered the ANA test without knowing this data, this can be the start of (in some cases) years of diagnostic limbo for patients. By the time they are finally retested for ANA by the more comprehensive IFA method, their symptoms will have progressed and may be more difficult to treat.
Ironically, had the same ANA plus reflex antibody panel been ordered 15 years ago, the initial ANA test would have been done by IFA, yielding a significantly more accurate result. This raises a serious question as to whether or not modern scleroderma diagnosis is being compromised by using these new, less expensive, testing methods.
Unfortunately, almost no primary care physicians (and some rheumatologists as well) are aware of these methodological problems with ANA testing, especially about the potential for incorrect negative results. The American College of Rheumatology in a 2011 Position Paper discusses these problems and recommends that testing by IFA “should remain the gold standard for ANA testing.” While it is true that ELISA and Multiplex ANA testing usually is consistent with IFA ANA testing, if an initial ANA result done by ELISA or Multiplex testing is negative, it is very important that the test be re-run by IFA to confirm the negative results.
However, between 2% and 10% of patients (depending on the study) with systemic scleroderma symptoms are ANA negative, even when done by IFA. In some cases, this negative result is false. For example, there are several different ways that ANA by IFA testing is done, for example using human cells or rodent cells to perform the test. Generally, ANA testing by IFA is done using human cells in the US, but if IFA testing is instead done with other types of cells, and the patient has one of the common scleroderma antibody types (anti-centromere or ACA), there is a significant chance that this antibody will not be detected, and the overall ANA result will falsely be reported as negative.
In most cases, the ANA does change to positive over time. When this occurs, it makes diagnosis very tricky in many cases. This is discussed more below. It is worth noting that the ANA level is generally stable over time, and there is no evidence that the actual tested ANA level is correlated with disease severity.
So what does this mean for someone who is starting the process of potentially being diagnosed with scleroderma? Having just read this information above about ANA testing, you now may well know more about this very narrow topic than almost all primary care docs (and possibly some rheumatologists who don’t specialize in this disease and may not have learned about this research). Educating your doctor can be a very tricky thing to do, but in this case, it is very important to make sure that s/he knows about ANA testing methodology and can thus order the correct type of ANA test. How to approach this potentially difficult interaction with your physician is discussed a bit later in this document.
Diagnosis – Step 2: Specific Antibody Testing
Actually, in spite of the discussion above, it is perfectly fine for your physician to initially order an ANA test with additional antibody testing, as long as s/he is aware of what method is being used to do the ANA test. The reality is that most of the time, ANA testing done with any of the three methods will end up with the same result. It is common practice at most testing laboratories that if an ANA done by ELISA or Multiplex ends up with a positive result, the standard procedure is to re-run it by IFA to confirm the positive result, and also get the ANA titer (a measure of antibody activity) and then run a disease-specific antibody panel (discussed below). The problem is that the ANA by IFA is not done to confirm a negative result, and this can significantly delay diagnosis if the negative result is a false one.
Once a potential scleroderma patient shows a positive ANA, the next step in diagnosis is to test for specific antibodies that can be used to help determine which form of systemic scleroderma the patient has or may develop in the future. Note that often when a patient first comes in with just a few symptoms, a skilled primary care practitioner may pick up some red flags that suggest an autoimmune disease as one possible explanation for the patient’s symptoms. The main symptom (when present) that should suggest to the PCP that there is a possibility of an underlying autoimmune disease is late-onset Raynaud’s. If a female patient, in particular, develops Raynaud’s in her teens or early 20s, then usually this is Primary Raynaud’s, meaning there is probably no underlying disease triggering the symptom, and it will typically just be an annoyance to the patient. In a small number of cases, an underlying autoimmune disorder does eventually develop, but if the primary care practitioner sees a patient that first develops Raynaud’s after age 30, and there is no other direct explanation, e.g., medication side effect, construction worker with vibration damage, certain circulatory problems, then this should automatically trigger at least ANA testing as part of the diagnostic workup.
Once it is determined that the patient has a positive ANA result, then the next step in diagnosis is running an appropriate antibody panel to try to figure out what may be going on. This may not actually be a separate step since most physicians will order an ANA test with a reflex antibody panel, but if the ANA is run separately first (by the IFA method), then the physician will order the panel separately after getting the ANA results. This follow-on panel will be specific to that likely disease. For example, if the patient presents with the symptoms listed in the earlier example (Raynaud’s, heartburn, puffy fingers), the physician may realize that scleroderma is the likely diagnosis and order a scleroderma specific panel. However, in many cases, the symptoms are ambiguous enough so that the panel will include the most common antibodies that could be present in a variety of different autoimmune conditions, including lupus and Mixed Connective Tissue Disorder (MCTD), as well as scleroderma. Since the focus of the Scleroderma FAQ and this Guide is on systemic scleroderma, I am not including information and antibodies specific to other autoimmune disorders.
The majority of systemic scleroderma patients will test positive for one of three scleroderma related antibodies: anti-Scl-70 (Topoisomerase I) antibodies, anti-centromere (ACA) antibodies, or anti-RNA polymerase III antibodies. The anti-Scl-70 antibody is highly specific for one of the diffuse forms of systemic scleroderma, and the anti-centromere antibody is highly correlated with a limited scleroderma variant. Historically, only the anti-Scl-70 and the anti-centromere antibodies were strongly associated with the two general categories of systemic scleroderma: diffuse and limited. The anti-RNA polymerase III antibody is now recognized as a third major scleroderma-related antibody. Patients with anti-RNA polymerase III antibodies are considered to be in the diffuse category, but the specific symptom profile is different from the typical symptoms shown by patients with anti-Scl-70 antibodies.
In addition to these three main antibodies, a number of other antibodies have been associated with different subtypes of systemic scleroderma, although these other antibodies are detected much less frequently than the three main antibody types listed above and commercial testing for some of these antibodies is not currently widely available. The Scleroderma FAQ has detailed information about these three main antibodies as well as a number of other scleroderma related antibodies that are less common (see the Antibody section of the Scleroderma FAQ for more information).
Diagnosis – Step 3: Additional Clinical Testing
When you visit your physician to try to understand what is going on, your specific symptoms will also help the physician decide what additional testing needs to be done. Even if s/he isn’t sure what might be a diagnosis yet, in addition to starting ANA and specific antibody testing, your physician will often decide to run additional tests even before you get initial lab results. For example, if you come in to your office visit indicating that you are having shortness of breath or difficulty exercising, s/he may order additional tests, such as a pulmonary function test (PFT), or a Doppler echocardiogram, or a CAT scan, to try to figure out what is happening with your lungs. If you indicate that you are having a lot of heartburn or difficulty swallowing, s/he may refer you to a GI doctor for an upper endoscopy or esophageal manometry exam. S/he may also want to make sure that your kidneys are functioning OK and order lab tests that are designed to look at potential kidney problems. All of these potential tests are described in more detail in the Scleroderma FAQ.
However, even if you are having none of these symptoms, once your physician gets enough information back from lab test results combined with your initial symptoms, it is possible that s/he may order many of these same tests in any case since in early stages of scleroderma, you may have subtle changes going on internally that are not yet causing symptoms but can be more easily treated if detected early. Recommended / standard testing for patients recently diagnosed with scleroderma is discussed later in this Guide.
Diagnosis – Step 4: Putting It All Together
So now that your physician has run a series of lab tests, looked at your clinical symptoms, and done additional testing that s/he feels is needed, you should be able to get a definitive diagnosis – right? Unfortunately, in many cases, it is not that easy. Let’s look at the diagnostic criteria for scleroderma to start. Up until 2013, the diagnostic criteria for scleroderma were based on a standard that was adopted back in 1980. In 2013, the American College of Rheumatology and the corresponding European organization published a new set of research-based standards for formally diagnosing scleroderma. These new standards take into consideration a number of symptoms that are strongly associated with scleroderma, for example, degree of skin thickening (if any), Raynaud’s, nailbed capillary enlargement, the presence of any of the three main scleroderma-related antibodies mentioned above, etc. This definitely will help clinicians diagnose more patients with scleroderma, but these new standards were actually developed to make it easier to do scleroderma research, not to help clinicians diagnose patients with scleroderma. The research article that describes these new standards does mention this, but unless you actually read the detailed article (which almost no clinicians will do), they will not realize this. Because of the way the research was done when developing these new standards, many symptoms that are strongly associated with scleroderma are not included, for example, GI symptoms such as heartburn or difficulty swallowing, or even major symptoms such as scleroderma renal crisis (SRC).
The new diagnostic standard includes a simple-to-use point system where numeric point values are assigned to various clinical symptoms. If the total number of points is 9 or greater, then the patient meets the formal (research) criteria for scleroderma. This is explained in detail in the Scleroderma FAQ. However, even then, it doesn’t distinguish among the scleroderma subtypes (e.g., limited or diffuse). Also, if the clinician ignores additional symptoms, s/he may decide (incorrectly) that the patient doesn’t have scleroderma because their point total is less than 9.
Another diagnostic challenge is that lab test results are sometimes not at all clear, or the symptom profile doesn’t quite fit the “normal” pattern for scleroderma (or another autoimmune disease). It turns out that a small number of patients truly have symptoms of more than one autoimmune disease, e.g., some form of scleroderma combined with some variant of lupus. These “crossover” situations are very difficult to figure out and different clinicians, even specialists, may interpret the patient’s symptoms differently. The good news is that this is not very common. The bad news is that if you are one of the patients with ambiguous test results and symptoms, you may have a hard time getting an accurate diagnosis. However, in most cases the treatments used are based more on the symptoms than on the formal diagnosis, so ultimately it may not make that much difference in what treatments are suggested by the patient’s symptoms. Unfortunately, in the US in particular, health insurance companies often decide what treatments they are willing to cover based on the formal diagnosis.