Diagnostic Tests

RUO (and LDT) is an international convention used to designate in-vitro diagnostic tests that have yet to be approved by regulatory agencies (FDA, Health Canada, etc). Some of these tests are in the process of regulatory review and these designations are commonly used by major labs and kit manufacturers. The test result reports of RUO / LDT designated tests are required to bear those designations so the clinician is aware that the test is not yet approved. However, this has no bearing on, and the designation is not an indication of, the clinical value of the tests/results.

Intravenous immunoglobulin (IVIG) therapy is a commonly used treatment for various autoimmune and inflammatory diseases. IVIG is a preparation of pooled human immunoglobulin G (IgG) derived from thousands of blood donors. Natural antibodies and natural autoantibodies are prominent in these preparations. While IVIG therapy has shown significant benefits in managing some autoimmune/autoinflammatory conditions, it is important to consider its potential effects on autoantibody and inflammatory disease lab tests.
Autoantibody testing plays a crucial role in diagnosing and monitoring autoimmune diseases such as systemic lupus erythematosus (SLE), rheumatoid arthritis, autoimmune neurological diseases, and many others. IVIG therapy can affect results in several ways:

• IVIG contains a wide range of antibodies that can compete with patient-derived autoantibodies during testing. This competition may lead to lower detectable levels of autoantibodies, potentially resulting in false negative results.
• IVIG preparations can also contain certain autoantibodies. This may lead to detectable levels of autoantibodies, potentially resulting in false positive results.
• Temporary suppression of autoantibody production: IVIG therapy can transiently suppress the production of autoantibodies, particularly in patients with active autoimmune diseases.
• This effect may lead to a decrease in autoantibody levels. If autoantibody levels(titers) are being used to follow-the disease course (remission or flares) this can be helpful feature to gauge effective response of IVIG.
• Interference with Specific Autoantibody Assays: Some autoantibody assays utilize techniques that may be affected by the presence of IVIG. For example, enzyme-linked immunosorbent assays (ELISAs) may exhibit non-specific binding or interference due to the high IgG concentration in IVIG preparations. Laboratories should be advised if a patient has received IVIG treatment so that methods can be used to minimize false positive or false negative results.
• Transient impact on Other  Inflammatory Markers: IVIG therapy can transiently affect various inflammatory markers, including acute phase reactants (i.e., CRP, ESR), cytokines, chemokines, and adhesion molecules. These changes may be related to the immunomodulatory effects of IVIG and can result in factitious fluctuations of the laboratory test results. It is important to consider the timing of patient sampling when interpreting these markers.
• Interference with Immunological Assays: IVIG contains a variety of immunoglobulins, which may interfere with specific immunological assays used to measure cytokines, chemokines, or other inflammatory markers.

SUMMARY: IVIG has a complex effect on autoantibody and related serological biomarker lab test results. Healthcare professionals should be aware of these effects to interpret test results accurately and make informed clinical decisions. Close collaboration between clinicians and laboratory specialists is essential to navigate the challenges posed by IVIG therapy and ensure optimal patient care in the context of autoimmune and inflammatory diseases.

RECOMMENDATION: If the patient is already receiving IVIG and there is a need to do autoantibody or related biomarker testing, it is best to draw the serum or plasma sample 1-2 days PRIOR to the next IVIG infusion. This can be a helpful approach to determining the impact and efficacy of IVIG in reducing autoantibodies and other molecules that are considered pathogenic that may be correlated with the clinical course (i.e., remission or flares) of the disease.

There is often a correlation between the ANA pattern and the presence of anti-DNA and autoantibodies to other intracellular autoantigens. Identification of the staining pattern is useful for the laboratory because it may influence the search for the most appropriate autoantibodies by disease specific autoantibody profiles or other more specific tests. For example, in the presence of a cytoplasmic or nuclear dot type of fluorescence, an immunoassay that includes the cytoplasmic antigens Jo-1, M2/PDC (mitochondria), ribosomal P, EEA1, GW Bodies or the Sp-100 autoantigens, may be indicated (3). In the presence of a homogenous pattern, a search for dsDNA, histone or chromatin antibodies may be indicated. Anti-nucleolar patterns remain one of the main challenges for the clinical laboratory because it is difficult using current technologies to identify the target antigens (fibrillarin, B23, PM/Scl, Pol I/III, Th/To and others) (4). However, when an anti-centromere pattern is present, confirmation is usually not necessary. More recently, there has been attention to the dense fine speckled (DFS) pattern and evidence indicating that patients with ‘monospecific’ anti-DFS antibodies DO NOT have an autoantibody associated rheumatic disease (5).

In general, the screening HEp-2 IFA titer does not correlate with clinical characteristics such as disease activity or flares, and therefore is not a particularly useful parameter for following the course of the disease or estimating the efficacy of therapy (10;11). It should be emphasized that this conclusion is not based on careful prospective laboratory studies using standardized tests on advanced diagnostic platforms or in defined indices of clinical disease (e.g. SLEDAI, SLAM). In general, titers of Hep-2 IFA may fluctuate over time, and the antibodies tend to be detectable in phases both of disease activity and remission (12;13), although there are reported exceptions. One exception may be the presence of high levels of anti-U1-RNP antibodies that are characteristic of mixed connective tissue disease (14).

Another exception is related to evidence that anti-dsDNA antibody levels often correlate with certain clinical features, e.g. lupus nephritis, and its determination is obligatory in the diagnostic work-up of SLE patients and the follow-up of nephritic cases (15;16). However, it is appreciated that some assays for anti-dsDNA detection are better than others in measuring clinically important shifts in antibody levels.

The HEp-2 IFA is the preferred screening method for detecting autoantibodies in human systemic autoimmune rheumatic diseases (SARD) and several other autoimmune conditions (i.e. primary biliary cirrhosis, autoimmune liver diseases, juvenile arthritis at risk of uveitis). In SARD the ANA has been referred to as the ‘gold standard’ for ANA screening (1). However, given the apparent low levels of some autoantigens in the HEp-2 cell substrates, such as the SSA/Ro, Jo-1 and ribosomal P, the test may have a negative result even when these autoantibodies are present (2). Therefore, if clinical findings are highly suggestive of Systemic Lupus, Polymyositis (Autoimmune Inflammatory Myopathy), Sjögren’s syndrome, Scleroderma or other systemic autoimmune conditions, the search for specific autoantibodies is most efficiently done by ordering a disease-specific profile, especially if the ANA result is negative.

Yes, when possible, it is important to try and identify specific antibody targets.  There is a wide variety of known autoantibody targets in systemic rheumatic diseases and this is expanding at a rapid pace (7-9). The HEp-2 IFA screening test is able to reveal more than 100 different types of autoantibodies (1), only a portion of which have a validated clinical association and only about 30-40 of these can be revealed by routine laboratory assays. From a cost-benefit point of view, therefore, it is not possible to detect the target specificity in all positive ANA cases. There is international consensus that to bring clinical value to HEp-2 IFA it should be reflexed testing to a disease-specific solid-phase assay.

The presence of high titer IgG autoantibodies and their persistence over time is characteristic of several autoimmune rheumatic diseases, such as SLE, scleroderma, Sjögren’s syndrome, mixed connective tissue disease and autoimmune liver disease (PBC, autoimmune hepatitis). High titer autoantibodies should not be regarded as epiphenomenona of infection or inflammation. However, autoantibodies at low titers (<1:80) may be present in patients with various non-autoimmune diseases (viral and bacterial infections, neoplasia, etc.), in relatives of patients with autoimmune diseases and in apparently healthy subjects MitogenDx has set a fixed cut-off for positivity at a titer of 1:80 to decrease the percentage of false positives. A large multicenter study has shown that ANA without any clinical significance may be found in 30% of healthy subjects at a titer of 1:40 and in 5% at a titer of 1: 160 (6).

Both tests use basically the same technology and assay – an indirect immunofluoresence assay (IFA) on HEp-2 cell substrates. The main difference is that many labs that perform the anti-nuclear antibody (ANA) test only report antibodies that react with the cell nucleus, while ignoring a wide spectrum of clinically-relevant autoantibodies that react with the cytoplasm and mitotic or cell cycle targets. Hence, the term ANA is restrictive, and the term anti-cell/cellular antibodies is more comprehensive and complete.

MitogenDx does not recommend repeating a Hep-2 IFA or ENA test until at least 6 months has passed from a previous blood draw and subsequent test.  However, if there is a compelling clinical reason an additional ENA or HEp-2 IFA test may be of insightful.  Repeated ANA and autoantibody profile tests are most useful in the diagnostic phase of patient evaluations (i.e. sera with initially negative or low titer positive ANA from a patient with a clinically defined systemic autoimmune disease). A repeat ANA or disease specific autoantibody profile is not indicated unless a change in the clinical picture raises the suspicion of a change in the underlying disease presentation or the appearance of another associated rheumatic disease (e.g. secondary Sjögren’s syndrome, secondary anti-phospholipid syndrome or an overlap syndrome, vasculitis, sudden appearance of Raynaud’s phenomenon).