The EUROLINE portfolio for autoimmune liver diseases (AILD) has been extended with the antigen F-actin and now features the DL 1300-9G with 10 antigens for the determination of AILD-related autoantibodies.
The EUROLINE Autoimmune Inflammatory Myopathies Profile has been expanded and now includes 20 antigens for detection and differentiation of autoantibodies that occur in idiopathic inflammatory myopathies (IIM).
EUROIMMUN has been creating innovative solutions for immunofluorescence diagnostics for over thirty years. Continuing this long tradition, the company has over the last year introduced further groundbreaking products to increase the efficiency and standardization of indirect immunofluorescence assay (IFA) analyses. CLI talked to Dr Panagiotis Grypiotis, Head of Product Management Autoimmune Diagnostics, Endocrinology/Biologics and Automation […]
A thorough understanding of the immune response to SARS-CoV-2 is necessary for the evaluation of the efficacy of vaccines and vaccine candidates against this virus. This article discusses how some parts of the humoral and cellular immune responses can be analysed to improve our understanding of how the body reacts to this virus.
Systemic vasculitides are a group of inflammatory idiopathic clinical syndromes usually classified by the size of the vessels being affected. Among them, the small vessels vasculitides show clear associations with the presence in the patients sera of antibodies directed against cytoplasmic antigens of neutrophils (ANCA). Thus, circulating antibodies against neutrophil cytoplasm is the most characteristic feature of the serological profile in patients with autoimmune vasculitides, as Wegener’s granulomatosis, microscopic polyangiitis and Churg-Strauss syndrome, the so-called anti-neutrophil cytoplasmic antibody-associated systemic vasculitides, or ANCA-associated vasculitis (AAV). This group of diseases show a mean prevalence of 20 per million inhabitants, with a higher rate in the age group of individuals older than 70.
by Dr Petraki Munujos
There are typically two laboratory techniques for ANCA detection, indirect immunofluorescence (IIF) and enzyme-linked immunosorbent assay (ELISA), that are meant to be used in a combined form: the sera showing positive fluorescence in the IIF assay are tested by means of ELISA to confirm the specificity of the antibodies directed against either proteinase 3 (PR3) or myeloperoxidase (MPO).
The classical terms used to describe the fluorescent patterns observed in the IIF test on human neutrophil granulocytes, C-ANCA (cytoplasm) and P-ANCA (perinuclear), refer to antibodies directed to antigenic targets located in the primary granules of neutrophils, like PR3, MPO, lactoferrin, catalase, enolase, elastase or human lysosomal membrane glycoprotein (h-lamp2), among others. In addition, there are also the so-called atypical patterns due to other antigenic targets different from PR3 and MPO or to a combination of specifities. The following are the four fluorescent patterns that can be observed with IIF on human neutrophils :
• C-ANCA, showing granular cytoplasmic neutrophil fluorescence with central interlobular accentuation. It corresponds in most of the cases to PR3 specificity.
• P-ANCA, with perinuclear neutrophil staining, often with nuclear extension, mostly corresponding to MPO specificity.
• ANCA-atypical or X-ANCA. The pattern is a mix of cytoplasmic and perinuclear rim-like fluorescence, and it is due to multiple specificities.
• C-ANCA-atypical. The cytoplasmic fluorescence is homogeneous, with no interlobular accentuation. Mainly due to the reaction of the antibodies with BPI (bactericidal/permeability-increasing protein).
However, it is important to keep in mind that the antigenic specificities corresponding to the fluorescent patterns observed are not clear, and that the assumption of C-ANCA being equivalent to PR3 and P-ANCA, to MPO is not correct. The positive results obtained by IIF must always be confirmed by specific ELISA tests.
A principle based on an artifact
Both PR3 and MPO are lysosomal enzymes that can be found in azurophilic granules in the cytoplasm of human neutrophilic granulocytes. The ethanol fixation of the neutrophil makes the positively charged proteins from the cytoplasm migrate towards the negatively charged nuclear membrane [figure 1]. As a result of this treatment, myeloperoxidase molecules in ethanol-fixed neutrophils locate in the perinuclear area instead of the cytoplasm. This circumstance makes it possible to distinguish the antibodies reacting with MPO from those reacting with PR3, since differentiated stained structures can be observed through the microscope [figure 2].
Laboratory detection of ANCA
As mentioned before, a sample analysed for the presence of ANCA by indirect immunofluorescence can show up to 4 different staining patterns, but C-ANCA and P-ANCA are by far the most commonly reported type of results. Since the correspondence with a target antigen is not unique for any of the stainings, sera containing ANCA should be tested in ELISA at least for both PR3- and MPO-ANCA. While the C-ANCA patterns clearly stain cytoplasmic components of the neutrophil, the P-ANCA pattern may be due to either cytoplasmic or nuclear targets, since distinguishing a cytoplasmic perinuclear staining from an homogeneous nuclear staining is unlikely. Consequently, sera containing P-ANCA should be further tested to discriminate those due to antinuclear antibodies (ANA) from those with antibodies directed to cytoplasmic antigens like myeloperoxidase. There have been classically two approaches for this purpose, consisting in testing the P-ANCA sera either to confirm the perinuclear localization of the antigenic target or to detect the presence of antibodies directed to nuclear structures like DNA or Histones. The first option is based on the use of neutrophils that have not been fixed with ethanol, therefore, the myeloperoxidase molecules have not migrated towards the perinuclear region of the cell. These neutrophil slides are fixed with formalin instead, allowing the myeloperoxidase to remain in its original cytoplasmic location. As a result, a serum containing anti-myeloperoxidase antibodies will show P-ANCA staining pattern on ethanol-fixed neutrophils and C-ANCA staining pattern on formalin-fixed neutrophils [Figure 3]. On the other hand, a serum containing ANA will show P-ANCA staining pattern on ethanol-fixed neutrophils and nuclear homogeneous staining pattern, often of weak intensity, or negative staining on formalin-fixed neutrophils [Figure 3].
The second approach to discriminate true P-ANCA sera from those containing antinuclear antibodies consists in testing for the presence of such antibodies, basically on HEp2 cells . This option may represent an additional cost, since the result only reveals the presence or absence of ANA but does not show direct evidences of the presence of antibodies directed against the cytoplasm of neutrophil. Some laboratories test in parallel both parameters, ANA and ANCA in a separate analysis. There have been some attempts to combine both ANA and ANCA tests in the same substrate, i.e., by mixing neutrophils and lymphocytes in the cellular preparation, but with controversial results due presumably to some incompatibility between the respective manufacturing procedures.
Besides the two typical patterns C-ANCA and P-ANCA, an unusual mixture of both staining patterns can be found, called atypical, or X-ANCA. On the other hand, not all myeloperoxidase molecules migrate to the perinuclear rim after ethanol fixation, and a “hybrid pattern” of both cytoplasmic and perinuclear staining can be observed and due to incomplete relocation of the antigen. Other antigens such as lactoferrin, elastase, BPI, cathepsin G, lysozyme, h-lamp-2 and enolase usually show X-ANCA pattern.
X-ANCA pattern (or atypical P-ANCA) is the result of the autoantibody binding mainly to the proteins cathepsin G, lysozyme and lactoferrin. These autoantibodies are not found in patients with Wegener’s granulomatosis, microscopic polyangiitis or Churg-Strauss syndrome, but are more often associated with inflammatory bowel diseases. Such proteins are released from the neutrophil specific granules and locate in the periphery of the nuclei after ethanol, acetone or formalin treatment . It is remarkable that the mentioned antigens do not show a different distribution within the cell as a result of the fixative used, contrary to what happens with MPO. This fact helps to distinguish the antibodies reacting against MPO (P-ANCA with ethanol, C-ANCA with formalin) from those reacting to cathepsin G, lysozyme or lactoferrin (P-ANCA regardless the fixative used). (Figure 4). P-ANCA can be distinguished from atypical P-ANCA by confocal microscopy in that the atypical p-ANCA show multiple intranuclear fluorescent invaginations of the multilobulated neutrophil nuclear envelope. However, this feature is very difficult to observe with a conventional microscope .
As mentioned, the perinuclear nature of some ANCA reactions is an artifact due to the ethanol fixation of neutrophils, causing a redistribution of the cytoplasmic granules around the nucleus. However, the atypical P-ANCA or X-ANCA reaction observed in patients with inflammatory bowel diseases like ulcerative colitis (UC) has been found not to be similarly affected, since the corresponding antigens have a nuclear localization. The comparison of IIF patterns of sera tested on different fixed cells may be useful to distinguish vasculitis-related P-ANCA versus ANA and vasculitis-related P-ANCA versus UC-related P-ANCA. On the other hand, methanol fixation seems to provide a brighter staining on X-ANCA patterns, as compared with ethanol-fixed neutrophils. However, some MPO positive sera become negative with this fixative, as compared with ethanol fixation, whereas no such effect is observed with PR3-ANCA and ANA positive sera .
ANCA are much more common in UC than in Crohn’s disease (CrD). ANCA testing alone does not distinguish between the two diseases, but the combination of ANCA test by IIF and anti–Saccharomyces cerevisiae antibody (ASCA) test by ELISA often aids their differentiation. Patients with UC are more likely to have P-ANCA–positive and ASCA-negative results, and those with CrD are more likely to have P-ANCA–negative and ASCA-positive results. Also, UC patients with moderate and severe disease usually have higher titres of ANCA than patients in remission or with mild disease . Due to the difficulties in distinguishing X-ANCA pattern from P-ANCA with the microscope, the determination of the final ANCA autoantigen specificity is often performed by means of ELISA assays using the corresponding specific antigen as microplate covering reagent.
Harmonization in ANCA testing
As a semi quantitative technique, indirect immunofluorescence results are expressed as low, moderate and strong positive, or negative, based on the analyst’s skills and interpretation. More than titrating the strong positive samples, what the international committees of experts recommend is to run additional tests to check the specificity by means of ELISA . Issues like the initial dilution, the choice of substrates or the sequence of tests to be performed are key aspects to further advance towards a better understanding of the laboratory data.
One of the goals of any organization devoted to standardization in the clinical laboratory is to establish harmonized testing algorithms. The European Autoantibody Standardization Initiative (EASI) has promoted several studies in that sense. Internationally standardized algorithms used by the different laboratories would contribute to the rational use of laboratory tests and would lead to a more efficient usage of the laboratory tests. Figure 5 shows the algorithm proposed by Conrad and colleagues faced with the suspicion of a systemic ANCA-associated vasculitis.
The International Consensus Statement advocates screening by IIF and confirmation of IIF positivity in PR3-ANCA and MPO-ANCA ELISAs , and this is a good example of the efforts addressed towards harmonization. However, many others aspects regarding not only the testing, but the manufacturing processes of IVD immunofluorescence reagents still need to be taken care of in terms of standardization.
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Petraki Munujos, PhD
Molecular allergology enables quantification of IgE antibodies to single allergen protein components at the molecular level. This helps the clinician establish the cause of allergic sensitisation, evaluate the risk for severe allergic reactions and improve patient management. New tests and technologies enable the laboratory to assist in an efficient manner.
by Dr Magnus Borres
For quite some time there has been scientific interest in the individual proteins contained in an allergy source, e.g. a pollen, foodstuff or animal fur. One of the first food allergen components, Gad c 1 from cod, was purified as early as the late sixties . The term Component Resolved Diagnostics was introduced in 1999 . Further developments, such as the production of recombinant allergen components and the use of microarray technology, have resulted in novel practical tools for the clinician and the laboratory. The term molecular allergology is now used to describe this new breakthrough science.
There is currently great interest in this area since several research studies have shown that molecular allergy diagnostics can result in:
- a more precise diagnosis of the allergic sensitisation by identification of specific versus cross-reactive components
- improved risk assessment by identification of components linked to a high- versus low risk of inducing severe, systemic reaction; and
- changed indications for immunotherapy or clinical management.
Allergen sources are complex
All allergen sources contain a number of different proteins, some of which can cause allergy. Each allergen component usually has several epitopes, the actual three-dimensional binding site for the corresponding antibody. Some allergen components are unique markers for a specific allergen source. Others have protein structures common to widely different species. In such cases IgE-sensitisation may be due to cross-reactivity with proteins with similar epitopes [Figure 1].
A positive extract-based IgE test does not give any information about which of the many components in the extract triggered the IgE response. Such information is of great value both for the clinician and the patient – is it a component that is present in many different allergen sources (cross-reactive sensitisation), or is it a species specific (primary) sensitisation? Furthermore, some components are prone to induce severe reactions while others cause sensitisation without clinical reactions. The stability of the component to heating and digestion also varies, and which is of great significance in food allergy.
Thanks to molecular allergology there are now good tools available for quantifying IgE sensitisation to allergen components, both tests for single components and microarray-based tests giving answers to 100+ components from a small amount of blood sample.
Single component tests
Allergen components are given designations based on the Latin family name of the species, e.g. Ara h 1 stands for the first allergen component of peanut, Arachis hypogea.
Tests for IgE antibodies to single allergen components are based on proteins purified from their natural source, or produced via recombinant techniques. Such single component tests provide high-quality, accurate, quantitative results and are therefore an essential tool for the allergy physician. They are especially useful for investigating allergy to one or a few suspected defined sources. The most comprehensive range of single component tests, ImmunoCAP Allergen Components (ThermoFisher Scientific, formerly Phadia AB, Uppsala, Sweden) contains over 90 allergen components.
Microarray or biochip technology makes it possible to simultaneously assay a large number of allergen components in a minute amount of patient sample. ImmunoCAP ISAC (ThermoFisher Scientific, formerly Phadia AB, Uppsala, Sweden) is a miniaturised immunoassay platform where allergen components are immobilised in a microarray. This enables simultaneous measurements of IgE antibodies to a fixed panel of 112 components from 51 allergen sources, using only 30 µl of serum or plasma. The allergen components are spotted in triplets and covalently immobilised to a polymer coated slide. Each slide contains four microarrays, giving results for four different samples per slide [Figure 2].
The assay consists of two steps. In the first, IgE antibodies from the patient sample bind to the immobilised allergen components. In the second, allergen-bound IgE antibodies are detected by a fluorescence-labelled anti-IgE antibody. The total assay time, including washing and incubation steps, is less than four hours.
The fluorescence is measured with a laser scanner and analysed by software that calculates the IgE results semi-quantitatively for each allergen component. The IgE concentration is measured in arbitrary units, so-called ISUs (ISAC Standardised Units), and these values are divided into four classes – negative, low, intermediate and high. This biochip technology provides a highly advanced tool for revealing the patient’s IgE antibody profile in an efficient manner.
Molecular allergology helps improve the diagnosis
The diagnosis of IgE-mediated allergic diseases is based on the clinical history and sensitisation confirmed by an allergy test. In some cases challenge tests are also performed to confirm the allergy diagnosis. For several years extract-based in vitro allergy tests have been available that give accurate and reproducible results. However, they have limitations in the sense that they are unable to identify the IgE-triggering molecule. Through molecular allergology the clinician now has new tools at his disposal in the form of single component tests and microarray-based biochips that can provide valuable help in the diagnosis and management of patients with various allergic symptoms .
Clinical usefulness of single components
An example of the clinical utility of molecular allergology may concern a child being investigated for suspected allergy to peanut. Peanut is the most common foodstuff associated with fatal allergic reactions in the Western world. The symptoms on ingestion of peanut may vary from mild reactions such as urticaria and oral allergy syndrome (OAS) to respiratory distress and severe systemic reactions such as anaphylactic shock.
A positive result from an IgE test based on peanut extract has a low predictive value, as many sensitised individuals are, in fact, tolerant to peanut. In some regions as many as two thirds of the peanut sensitised patients are actually peanut tolerant . The reason for this is due to cross-reactive IgE antibodies with low clinical significance, e.g. antibodies induced by proteins in pollens (PR-10 proteins) that cross-react with homologous proteins in the peanut.
Now molecular allergology offers new tools for investigating the nature of the peanut sensitisation. Tests for five clinically relevant peanut components are available. Three different seed storage proteins, Ara h 1, Ara h 2 and Ara h 3, are all important allergens and responsible for primary sensitisation to peanut. Ara h 2 is particularly considered a risk marker for severe allergic reactions. Sensitisation to more than one of these allergens is also a stronger indication of more serious reactions than sensitisation to just one of them. A fourth component, Ara h 8 (a PR-10 protein), is a homologue to the birch pollen component Bet v 1 and thus a marker for sensitisation to pollen with low clinical relevance in peanut allergy.
Finally, Ara h 9 is a so-called lipid transfer protein (LTP). IgE antibodies to Ara h 9 are often linked to OAS, but also to systemic and serious reactions in southern Europe. This may be an indicator of primary sensitisation to peach and other fruits containing LTPs.
A child tested for peanut allergy should be managed very differently depending on the outcome of the tests. If the sensitisation is linked to Ara h 8 the child is not at risk of severe anaphylactic shock, information of great value to the patient. On the other hand, if the sensitisation is to one or several of the storage proteins it is essential that the child always carries injectable adrenaline.
In a similar fashion, molecular allergology has brought new diagnostic tools enabling the investigation of the sources of IgE sensitisation to a number of other foodstuffs such as tree nuts, egg, milk, wheat, fish and soy, and also to furred animals, insect venoms, mites and pollen.
The selection of patients for allergen-specific immunotherapy (SIT) is another area where knowledge of the source of sensitisation is of great value. Accurate prescription of SIT depends on the exact identification of the disease-eliciting allergens. In a recent study by Sastre et al. it was shown that the use of molecular allergology resulted in changed SIT prescriptions in as many as 54% of the patients, compared to traditional diagnostic methods .
Challenge tests have long been considered the gold standard for diagnosing food allergy. However, this is a resource-demanding method and is not without risk to the patient. As single component testing already constitutes a valuable tool for investigating whether the patient is suitable for food challenge or not, it is also a fair question to ask if molecular allergology can lead to in vitro tests that could replace or reduce the cumbersome food challenges.
The clinical advantages of biochips
The microarray-based ImmunoCAP ISAC is an efficient tool for establishing patient sensitisation profiles by simultaneous measurements of IgE antibodies to a fixed panel of 112 components.
ImmunoCAP ISAC is especially useful with “problematic” patients, e.g. patients with inconsistent or diffuse symptoms and case histories, patients not responding to treatment, and multi-sensitised patients where standard tests give complex results. The biochip test gives comprehensive information about the patient’s sensitisation profile , making it possible to distinguish between primary and cross-reacting sensitisers. It also reveals potential risks for reactions of various types and unexpected sensitisation.
By establishing the patient’s sensitisation profile, sensitisation can be detected at an early stage, before clinical symptoms have developed, thus enabling a better prognosis and the initiation of preventive measures .
True, interpreting 112 allergen component test results per patient may be challenging for the clinician, but PC-based intelligent support for interpretation is available.
Molecular allergology provides laboratories with a novel test portfolio more and more in demand by clinicians. This new science enables a more individual-based approach to allergy diagnosis and the clinical management of allergic patients.
Single component tests are a good tool for quantitative detection of IgE antibodies to the individual proteins of allergen sources, making it possible to determine if IgE sensitisation is species specific or the result of cross-reactivity. This information is essential in order to assess the risk for reactions and to identify the right patients for immunotherapy.
Microarray-based tests furthermore makes it possible to simultaneously assay a large number of allergen components in a minute amount of patient sample, giving a broad spectrum of the patient’s IgE profile on the molecular level. This is especially useful in the management of multi-sensitised patients and patients with diffuse symptoms.
Molecular allergology enables the clinician to make better diagnoses and prognoses, prescribe more accurate treatment and offer better advice on avoidance. Further work may also bring the possibility to replace currently used diagnostic procedures that are resource-intensive, costly and potentially dangerous.
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Magnus Borres, MD, PhD, MPH
Pediatric Allergist, Karolinska University Hospital, Stockholm, Sweden
Medical Director, ImmunoDiagnostics, Thermo Fisher Scientific, Uppsala, Sweden
The HEp-2 immunofluorescence assay (IFA) for ANA screening is excellent for ruling out many connective tissue diseases, but a positive result seldom translates into a clinically meaningful diagnosis. A new automated, efficient, enzyme immunoassay for ANA screening provides reliable, objective information that can be applied clinically with confidence.
by James D. Peele, PhD
For laboratory directors, the immunofluorescence assay (IFA) using HEp-2 cells is one of the most commonly ordered tests for antinuclear antibody (ANA) screening. ANA screening has been considered, along with patient history and physical examination, as one of the diagnostic tools for systemic autoimmune rheumatic diseases (SARD) for decades . While the ANA screen is excellent for ruling out autoimmune diseases, possessing good sensitivity and negative predictive value (NPV), its specificity and positive predictive value (PPV) are less impressive. In the majority of cases, a positive ANA by IFA seldom translates into a clinically meaningful diagnosis. For example, 4 out of 5 positive HEp-2 results have no clinical relevance according to Peene and colleagues, who tested a large, consecutive cohort of serum samples . More recently, 90% of patients referred to a tertiary rheumatology clinic for a positive ANA had no evidence of ANA-associated rheumatic disease . Indeed, 31.7% of the normal population is ANA positive at a 1:40 serum dilution, 13% at 1:80, and 5% at 1:160 . The financial impact for such unnecessary follow-up testing or specialist referrals continues to negatively impact any healthcare system.
These truths pose a dilemma for laboratory directors, who experience HEp-2 ANA testing as a laborious manual operation, which is designated as highly complex and requires skilled technologists . In addition, consistency in performance of IFA testing and interpretation of results within and between labs is problematic, despite attempts at standardization. Interpreting titres and patterns can at times seem more art than science.
Using new ELISA-based technology (sometimes called EIA for enzyme immunoassay) to screen for multiple extractable nuclear antigen (ENA) markers, centromere and double-stranded DNA (dsDNA) has automated testing at Baptist Medical Center and greatly improved the PPV for our ANA screen. The most recent international recommendations for the diagnosis of SARD advise that a specific panel of laboratory tests be ran to include ANA, anti-dsDNA and anti-ENA antibodies,  which collectively make up what we commonly call our ANA screen. Our experience illustrates how evolving technology can work hand-in-hand with clinical expertise to improve the diagnostic process.
Comparing EIA with conventional ANA testing
The long tenure of HEp-2 ANA testing means that rheumatologists have grown accustomed to the test’s utility and to its limitations. The American College of Rheumatology (ACR) emphasizes that “A positive ANA test means autoantibodies are present. By itself, a positive ANA test does not indicate the presence of an autoimmune disease or the need for therapy” . This understanding may not be applied among primary care physicians, who often order the test before referral. Some specialists actively discourage the use of IFA as a screening tool in primary care because of its extremely low specificity and PPV .To bridge the gap between familiar HEp-2 ANA testing and new ANA screening technologies, ACR has recommended that new assays demonstrate the same or improved sensitivity and specificity compared to IFA. In searching the literature and conducting our own studies, we have found that EIA compares well to IFA for ANA screening.
Two studies demonstrate this comparison. The first evaluated 170 pre-selected serum samples from clinically defined patients by comparing the results of IFA testing against an EIA screening assay (EliA® Symphony, Thermo Fisher Scientific) . The results, presented in Table 1, demonstrate comparable performance and efficiency.
The second study examined 388 consecutive samples that were measured using HEp-2 IFA (1:160), an EIA screen and, if that were positive, single ENA differentiation antigens . The study found 84% agreement of results (Table 2), with IFA missing several clinically relevant dsDNA and Ro-negative samples and EIA missing some nucleolar patterns.
Our experience at Baptist Medical Center
We were aware of these studies before introducing the new ELISA-based technology into our laboratory. We also conducted our own testing, to assure our laboratory personnel and ordering physicians that others’ results could be replicated in our setting. Our testing was uniformly encouraging (Table 3). Most notably, the specificity and PPV of EIA testing were greatly improved compared with IFA, and without any appreciable loss in sensitivity.
Still, smooth implementation of any new diagnostic test requires an understanding of its clinical utility and of established practice patterns. Thus, we met with our rheumatologists to explain how the new EIA test compared to the IFA method they were using. Typically they order ANA testing after classifying patients according to established autoimmune disease criteria, as Vos and colleagues did in a study of 328 serum samples (Table 4) . In that study under conditions of daily clinical practice, a similar percentage of patients was found by both methods in each subgroup. Overall, the differences between IFA and EIA were small.
We told the rheumatologists that a positive result on the new ANA-EIA screen indicated the presence of one or more clinically significant antibodies (dsDNA, Sm, U1RNP (RNP 70, A, C), SSA (60 kDa and 52 kDa), SSB, Scl-70, Jo-1, or centromere). And we agreed to offer the HEp-2 IFA methodology on request, should any clinicians need it. We also asked, “If you knew that DNA, centromere and ENA markers were negative, would you still need a pattern and titre?” This led to a discussion of the high number of false-positive results (approximately 25%-30%)  found with the traditional IFA-ANA method. Many of these false positives could be associated with the dense fine speckle 70 (DFS70) antigen pattern, which has been found in 33% of ANA-positive healthy individuals but not in ANA-positive sera from patients with SARD .
We also informed physicians of the method switch from IFA to EIA screening for our ANA screen, and of the expansion of specific ENA testing for any follow-up testing needs. For positive ANA screen tests, follow-up identification of specific ENA markers can add predictive diagnostic value in their specialist practice  A list of frequently asked questions (FAQs) when comparing the two methods was provided to our clinicians as part of this implementation process. These FAQs explained how results would be reported, and alerted physicians to the new testing schedules, including same-day results for tests submitted early in the day. Moreover, we assured them that unlike ANA by IFA, a positive result on an ANA-EIA screen would be positive for an ENA marker, centromere or dsDNA >87% of the time. These steps resulted in a positive reception. We’ve had only a few requests for concurrent IFA testing since we switched over a year ago, and the transition went far better than expected.
Improving laboratory efficiency and clinical utility
Our experience bodes well for the laboratory, physicians and patients. From the laboratory perspective, we can deploy skilled technologists more strategically because of the automation of EIA testing. We currently use less labour to run ANA and dsDNA testing daily than we did for once-a-week testing, and ENA testing has been increased to two or three times per week. We also added CCP and rheumatoid factor testing without requiring any additional labour. Automation has streamlined workflow efficiency, as a technologist can walk away from the Phadia® 250, whereas the manual process required constant attention for a full day. We have also eliminated the complexity and subjectivity of interpreting IFA patterns. Only two technologists were proficient with the complicated manual testing, but more of our personnel possess the skills necessary to operate the new equipment. Automated EIA testing not only provides excellent quality assurance, but also screens for specific, clinically relevant autoantibodies, unlike the broad range of antinuclear antibodies detected by HEp-2 IFA.
From the clinical perspective, providing more specific results in a timely manner allows physicians to focus on the correct diagnosis and treatment without delay because of improved clinical specificity and PPV. False-positive test results from IFA can lead to overdiagnosis and misdiagnosis. Both carry the risk of inappropriate treatment with potent medications, unnecessary referrals, additional laboratory testing, and higher healthcare utilization and costs . Meanwhile, patients may suffer both emotionally and economically. By contrast, we have found that automated and efficient EIA testing for ANA provides reliable, objective information that can be applied clinically with confidence.
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3. Abeles AM, Abeles M. The clinical utility of a positive antinuclear antibody test result. Am J Med 2013;126(4):342-348.
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James D. Peele, PhD
Director of Clinical Chemistry
Baptist Medical Center
Autoantibodies against MDA-5 are serologically important biomarkers because they are mainly detected in patients with amyopathic dermatomyositis complicated with rapidly progressive interstitial lung disease (ILD). Anti-MDA-5 antibodies are useful not only for diagnosis but possibly also for monitoring disease activity in ILD.
by Assoc. Prof. Y. Muro, Assoc. Prof. K. Sugiura and Prof. M. Akiyama
Dermatomyositis and autoantibodies against melanoma differentiation-associated gene 5
Idiopathic inflammatory myopathies (IIMs) are a group of systemic autoimmune diseases that include polymyositis (PM), dermatomyositis (DM) and inclusion body myopathies . Several myositis-specific autoantibodies (MSAs) are associated with certain clinical forms of IIMs, and they are useful tools for predicting the prognosis of IIMs. For example, patients that are positive for anti-transcriptional intermediary factor 1-gamma (TIF1-γ) antibodies are often complicated with internal malignancies, and patients that are positive for anti-melanoma differentiation-associated gene 5 (MDA-5) antibodies demonstrate rapid progressive interstitial lung disease (ILD) . Since these complicating diseases are life-threatening, the above antibodies are very important biomarkers.
Many recent studies have clarified new MSAs which are associated with clinical subsets of DM/PM. As MSAs are basically mutually exclusive, they also can be used to determine the disease subset. A subgroup of DM patients is known to have typical skin manifestations of DM but with little evidence of myositis, a condition known as clinically amyopathic DM (ADM). Initially, anti-MDA-5 antibodies were reported to be serological markers of clinically ADM with rapidly progressive ILD, especially in East Asia; they also are found in Caucasian patients with ADM complicated with ILD. A Japanese multicentre study found the 5-year survival for patients with anti-MDA-5 antibodies to be 56% .
Function of MDA-5
The MDA-5 gene was originally identified as an upregulated sequence in a cDNA subtraction library screening of terminally differentiating HO-1 human melanoma cells by combined treatment with IFN-β and mezerin . The innate immune system senses RNA virus infections through membrane-bound toll-like receptors or the cytoplasmic proteins RIG-I and MDA-5 . RIG-I and MDA-5 proteins comprise an amino-terminal caspase recruitment domain (CARD) and a carboxyl-terminal RNA helicase-domain. These sensors interact with a CARD-containing adaptor protein, IPS-1, located in the outer membrane of mitochondria after binding viral RNA. This interaction mediates recruitment and activation of protein kinases that phosphorylate the transcription IFN-regulatory factor 3, leading to the synthesis of type I IFN. RIG-I is essential for detecting infection by rhabdoviruses, influenza viruses, paramyxoviruses, and flaviviruses. Replication of these viruses leads to the production of RNAs with a 5’-triphosphate. In contrast, MDA-5 senses infection by picornaviruses and long-strand dsRNA. The mechanism for the discrimination of RNA ligands by RIG-I and MDA-5 is unknown. In a large screening of single-nucleotide polymorphisms associated with type 1 diabetes, a number of mutations in the MDA-5 gene were reported.
Discovery of anti-MDA-5 antibody
Anti-MDA-5 antibody was initially named anti-CADM-140 antibody before the recognized antigen was clarified . CADM is an abbreviation of ‘clinically amyopathic dermatomyositis’, and the molecular size on SDS-PAGE is 140 kDa, as shown by immunoprecipitation. In 2009 and 2010, two independent groups identified MDA-5 as its autoantigen by cDNA expression library immunoscreening or by peptide mass fingerprinting [7, 8]. The common clinical features of patients with anti-MDA-5 in both studies were those typical of ADM (i.e. dermatomyositis with scarce manifestation of myositis), frequent complication of ILD and poor prognosis.
Detection of anti-MDA-5 antibodies
Indirect immunofluorescence assay using HEp-2 cells as the substrate is the most commonly used routine test for the detection of anti-nuclear/cytoplasmic autoantibodies. However, anti-MDA-5 antibodies show weak cytoplasmic pattern or frequently negative results. Although radioisotope-based immunoprecipitation assays have tended to be used to detect anti-MDA-5 antibodies, such assays are not easy to perform. Sato et al. established an ELISA for anti-MDA-5 antibodies by using recombinant protein produced by a baculovirus expression system . To date, commercially available immunoassay kits that may clarify the unknown clinical significance of anti-MDA-5 across races or countries do not exist. It should be noted that anti-MDA-5 antibodies in patients do not react to RIG-I .
Longitudinal study of anti-MDA5
Systemic lupus erythematosus is also an autoimmune rheumatic disease that is characterized by a fluctuating disease course and a variety of autoantibodies. Many autoantibody specificities (SS-A/Ro, SS-B/La, Sm, U1-RNP) in lupus patients remain constant over time, whereas reactivity to dsDNA may fluctuate with disease activity, although the pattern of change differs with autoantibody specificity . We established a quantitative assay of antibody levels and monitored anti-MDA-5 autoantibodies during long-term follow-up periods in order to assess the long-term outcome of ADM patients with anti-MDA-5 antibodies . Our ELISA used biotinylated recombinant protein expressed by an in vitro transcription and translation system and streptavidin-coated plates. The levels of anti-MDA-5 were measured at different time points in 11 ADM patients who tested positive for anti-MDA-5 on their first visit (range of follow-up: 3 months to 16 years). At the stage of clinical remission of ADM+ILD, 6 of the patients received no medication and the 4 others received low-dose corticosteroid. One patient passed away due to rapidly progressive ILD. Surprisingly, the anti-MDA-5 antibodies disappeared in 9 of the patients and fell to just above the cut-off in 1 patient, whereas in the patient who died, the antibodies remained, as shown by ELISA.
Gono et al. showed that the decline index of the anti-MDA5 level after treatment was lower in patients who died than in patients who survived . We also recently came across an anti-MDA5-positive DM patient with ILD who showed a decrease in anti-MDA5 level before a fall in other biomarkers after therapy and favourable prognosis.
A 42-year-old Japanese man noticed erythema and papules on the dorsal surfaces of his hands in June and later erythema also on the face, neck and back. In July, he consulted a general practitioner, was suspected of having a collagen disease and was referred to our hospital. He had facial erythema and Gottron’s sign (i.e. erythema with slight scaling over the metacarpophalangeal and proximal interphalangeal finger joints). No muscle weakness was found, and the creatine kinase level was within the normal range. Although he noticed slight dyspnea upon exertion, interstitial changes to both lower-lung fields were seen by computed tomography. Our ELISA showed him to be positive for anti-MDA-5 antibodies, and various immunosuppressive agents were used for therapy (Fig. 1). He showed a decrease in anti-MDA-5 level before falls in other biomarkers (ferritin, SP-D) after intensive immunosuppressive therapy. He has now been under observation by our hospital on a minimum maintenance dose of prednisolone (5 mg/day).
Conclusions and future
In the previous studies [11, 12], anti-MDA-5 levels were higher in patients who later died than in patients who survived, but the difference in levels was not statistically significant. Although we agree that the anti-MDA-5 level is useful for the evaluation of response to treatment in ILD with ADM, it should be stressed that single-point evaluation of anti-MDA-5 level has limitations in predicting the prognosis of these patients. Other factors, such as the anti-MDA-5 isotype, in predicting the prognosis of these patients remain to be clarified. Commercially available immunoassay kits will be helpful for the investigation, as well as the monitoring, of each patient.
Not only is the anti-MDA-5 antibody a diagnostic marker of ADM+ILD, but it is also useful as an indicator for response to therapy. Considering that MDA-5 plays important roles in the innate immune system during RNA viral infections, an autoantigen/autoantibody system of MDA-5 should be linked to the pathogenesis of this disease condition. For example, MDA-5 was shown to be degraded in cells infected with different picornaviruses . Whether such cleavage might lead to autoimmune responses against MDA-5 (i.e. antigen-driven) needs investigation.
1. Mammen AL. Dermatomyositis and polymyositis: Clinical presentation, autoantibodies, and pathogenesis. Ann N Y Acad Sci. 2010; 1184: 134–153.
2. Hoshino K, Muro Y, Sugiura K, Tomita Y, et al. Anti-MDA5 and anti-TIF1-γ antibodies have clinical significance for patients with dermatomyositis. Rheumatology 2010; 49: 1726–1733.
3. Hamaguchi Y, Kuwana M, Hoshino K, Hasegawa M, et al. Clinical correlations with dermatomyositis-specific autoantibodies in adult Japanese patients with dermatomyositis: a multicenter cross-sectional study. Arch Dermatol. 2011: 147: 391–398.
4. Jiang H, Fisher P. Use of sensitive and efficient subtraction hybridization protocol for the identification of genes differentially regulated during the induction of differentiation in human melanoma cells. Mol Cell Different. 1993; 1: 285–299.
5. Barral PM, Sarkar D, Su Z-Z, Barber GN, et al. Functions of the cytoplasmic RNA sensors RIG-I and MDA-5: key regulators of innate immunity. Pharmacol Ther. 2009; 124: 219–234.
6. Sato S, Hirakata M, Kuwana M, Suwa A, et al. Autoantibodies to a 140-kd polypeptide, CADM-140, in Japanese patients with clinically amyopathic dermatomyositis. Arthritis Rheum. 2005; 52: 1571–1576.
7. Sato S, Hoshino K, Satoh T, Fujita T, et al. RNA helicase encoded by melanoma differentiation-associated gene 5 is a major autoantigen in patients with clinically amyopathic dermatomyositis: Association with rapidly progressive interstitial lung disease. Arthritis Rheum. 2009; 60: 2193–2200.
8. Nakashima R, Imura Y, Kobayashi S, Yukawa N, et al. The RIG-I-like receptor IFIH1/MDA5 is a dermatomyositis-specific autoantigen identified by the anti-CADM-140 antibody. Rheumatology (Oxford) 2010; 49: 433–440.
9. Tench CM, Isenberg DA. The variation in anti-ENA characteristics between different ethnic populations with systemic lupus erythematosus over a 10-year period. Lupus 2000; 9: 374–376.
10. Muro Y, Sugiura K, Hoshino K, Akiyama M. Disappearance of anti-MDA-5 autoantibodies in clinically amyopathic DM/interstitial lung disease during disease remission. Rheumatology (Oxford) 2012; 51: 800–804.
11. Gono T, Sato S, Kawaguchi Y, Kuwana M, et al. Anti-MDA5 antibody, ferritin and IL-18 are useful for the evaluation of response to treatment in interstitial lung disease with anti-MDA5 antibody-positive dermatomyositis. Rheumatology (Oxford) 2012; 51: 1563–1570.
12. Muro Y, Sugiura K, Akiyama M. Limitations of a single-point evaluation of anti-MDA5 antibody, ferritin, and IL-18 in predicting the prognosis of interstitial lung disease with anti-MDA5 antibody-positive dermatomyositis. Clin Rheumatol. 2013; 32: 395–398.
13. Barral PM, Morrison JM, Drahos J, Gupta P, et al. MDA-5 is cleaved in poliovirus-infected cells. J Virol. 2007; 81: 3677–3684.
Yoshinao Muro* MD, PhD; Kazumitsu Sugiura MD, PhD; and Masashi Akiyama MD, PhD
Division of Connective Tissue Disease and Autoimmunity, Department of Dermatology, Nagoya University Graduate School of Medicine, Nagoya, JAPAN
The detection of liver/kidney/microsomal antibodies (LKM) is one of the most common analytical procedures performed in the autoimmunity laboratory. Several techniques can be currently adopted that allow the detection of LKM in patient serum, such as ELISA, line immunoassay or indirect immunofluorescence. Far from being outdated, indirect immunofluorescence is the major method used for the screening of autoantibodies in autoimmune disease diagnosis. But LKM antibodies detection is one of these tests that require skilled laboratory technicians for an accurate pattern identification. This article gives a few tips that can be helpful for that purpose.
The detection of antibodies against microsomal antigens from liver and kidney, LKM (liver/kidney/microsomal), is an aid for the diagnosis of type II autoimmune hepatitis (Type II AIH). LKM antibodies belong mainly to the IgG isotype, and three different kinds can be distinguished: LKM-1 antibodies, considered as biomarkers of type II AIH; LKM-2 antibodies, which are present in cases of tienilic acid-induced hepatitis; and LKM-3 antibodies, found in some patients with chronic hepatitis D. LKM-1 antibodies can also be detected in patients of type I autoimmune hepatitis that show negative titres for antinuclear antibodies .
Cytochrom P450 II D6, the molecular target for LKM antibodies¸ is a 50kDA protein found in the cytoplasm of all hepatocytes and renal proximal tubular cells. This protein belongs to the microsomal enzymes family, localized in the endoplasmic reticulum membrane .
There are two important families of endoplasmic reticulum enzymes, the cytochroms and the UDP-glucuronosyl transferases (UGT). Corresponding to the characteristics of the antibodies directed to those enzymes are a certain number of related specifities (table 1).
Two distinct subtypes of autoimmune hepatitis, type I and type II autoimmune hepatitis, can be discriminated with the aid of the autoimmune serology: antinuclear (ANA) and anti-smooth muscle antibodies (ASMA) lead to the diagnosis of type I AIH, whereas LKM-1 antibodies assist in the diagnosis of type II AIH. The differing serological reactivities for the two types (ANA, ASMA versus LKM-1) are mutually exclusive, although in rare exceptions with ‘double positive’ serology, the clinical manifestations resemble those of type II AIH. Thus, LKM-1 antibodies are considered as biomarkers of type II AIH .
The disease usually starts in childhood and it often shows a severe progression, affecting more frequently women, like most autoimmune diseases. In the U.S.A., LKM-1 antibodies are common in pediatric patients and rare in adults. LKM-1 antibodies can be detected in certain patients with hepatitis C virus infections, not being affected by type II AIH .
Indirect immunofluorescence on rodent kidney and liver sections is the main technique used to test LKM-1 antibodies in the clinical laboratory. The typical fluorescent pattern observed consists in an intense staining of proximal renal tubules in kidney and intense cytoplasm staining of hepatocytes in liver (Figure 1). Due to the similiarity between the mitochondrial and the LKM-1 patterns, tests manufacturers usually supply slides containing kidney with cortex and medulla, liver and stomach in the same determination well to facilitate the interpretation of LKM antibodies.
A helpful indiction is the negativity of LKM antibodies in the renal medulla, composed only of distal tubules, whereas mitochondrial antibodies stain equally both, proximal and distal renal tubules (Table 2). Among the differential features, mitochondrial antibodies generally show a granular aspect, with glomeruli clearly visible and all tubules stained with certain intensity. On the contrary, LKM-1 antibodies show a non granular staining with glomeruli and distal tubules completely negative.
A part from indirect immunofluorescence on rat kidney and liver sections, ELISA and line immunoassays are techniques commonly used in the clinical laboratory for LKM antibodies determination. The antigen mostly immobilized in this kind of assay is the recombinant cytochrom P450 II D6, used as a capture molecule.
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2. Manns MP, Griffin KJ, Sullivan KF, Johnson EF. LKM-1 autoantibodies recognize a short linear sequence in P450IID6, a cytochrome P-450 monooxygenase. J Clin Invest 1991;88(4):1370-1378.
3. Strassbourg CP, Manns MP. Autoantibodies and autoantigens in autoimmune hepatitis. In: PD Berk, MP Manns, eds., Seminars in liver disease, 2002, volume 22, number 4: 339-351.
4. Vergani D, Alvarez F, Bianchi FB, Cançado ELR, Mackay IR, Manns MP, Nishioka M, Penner E. Liver autoimmune serology: a consensus statement from the committee for autoimmune serology of the International Autoimmune Hepatitis Group. J Hepatol 2004; 41: 677–683
5. Krawitt,E.L. Autoimmune hepatitis. N Engl J Med. 2006;354(1):54-66.
Petraki Munujos, PhD
The spectrum of autoimmune neurological syndromes has expanded substantially in the last fifteen years because of the discovery of novel anti-neuronal autoantibodies. Today’s autoantibody test portfolio includes over 32 different specificities associated with neurological diseases. Alongside classic anti-neuronal autoantibodies against intracellular targets such as Hu, Yo, Ri, etc are newly identified autoantibodies directed against cell surface antigens, for example glutamate receptors of type NMDA or AMPA, GABAB receptors, the voltage-gated potassium channel-associated proteins LGI1 and CASPR2, or DPPX. Since many of the autoantibodies occur only rarely, multiplex screening is favoured over selective or sequential testing to avoid diagnostic gaps. Anti-neuronal autoantibodies can be efficiently screened using multiplex indirect immunofluorescence tests (IIFT) comprising mosaics of tissue sections and monospecific recombinant-cell substrates. Immunoblots containing extensive panels of purified antigens are ideal for confirming antibody specificities.
Paraneoplastic neurological syndromes
Paraneoplastic neurological syndromes (PNS) are disorders of the central and peripheral nervous system that occur in direct relation to tumour development. However, they are not caused directly by the tumour or its metastases or by therapeutic side effects. The cells of the tumour express antigens that normally only occur in neurons. These induce the formation of specific antibodies, which then bind to the corresponding antigens in the nervous tissue and damage the neurons. PNS develop in around 15% of malignant diseases, occurring most frequently in small-cell lung carcinoma, neuroblastoma, thymoma, and cancers of the ovary, mamma, uterus and testis.
Classic onconeuronal antibodies in PNS include, for example, anti-Hu, -Yo, -Ri, -amphiphysin, -CV2, -Ma1, -Ma2/Ta, -recoverin, -PCA-2, -Zic4, -SOX1 and -DNER (Tr). These antibodies are directed predominantly against intracellular antigens, with the exception of anti-DNER which target a receptor (delta/notch-like epidermal growth factor-related receptor). If a positive result is obtained for PNS antibodies, the probability of a tumour is over 95%. Thus, if corresponding clinical symptoms are present and differential causes have been excluded, the detection of well-characterized anti-neuronal autoantibodies is considered sufficient to make a definitive diagnosis of PNS. Moreover, the antibodies may be detected up to five years before the tumour appears. Hence, in positive cases, PET scans should be performed on a regular basis to search for the tumour, which can then be excised at an early stage.
Stiff-person syndrome is a rare neurological disease which can be paraneoplastic or non-paraneoplastic in origin. The disease manifests with severe progressive muscle stiffness, typically in the spine and lower extremities. Paraneoplastic cases are associated with antibodies against amphiphysin. Non-paraneoplastic cases are characterized by autoantibodies against glutamate acid decarboxylase (GAD), which are found in 60-90% of patients. However, anti-GAD antibodies are not specific markers for stiff-person syndrome as they also occur in other neurological diseases and, in particular, in diabetes mellitus type I.
Autoimmune limbic encephalitis
Autoimmune limbic encephalitis encompasses a range of disorders manifesting with memory deficits, neuropsychiatric symptoms and epileptic seizures. A few years ago this condition was primarily attributed to classic paraneoplastic antibodies. However, in recent years several novel autoantibodies have been described as being associated with this condition. They differ from classic antibodies in that they are directed against target antigens on the cell surface of neurons, typically canal or receptor proteins. In some cases the autoantibodies are associated with malignancies, but in many cases no tumour is detected. Thus, the associated encephalitides are classified as facultative PNS. The autoantibodies are ascribed a direct pathogenic role. Binding of the autoantibodies to the corresponding membrane proteins interferes with synaptic signal transduction, resulting in neuropsychiatric deficits. A favourable prognosis for autoimmune limbic encephalitides is highly dependent on early diagnosis and intervention. The immediate initiation of immunotherapy and, in paraneoplastic cases, tumour removal helps to stabilize the patients and improve their overall outlook.
Cell surface antigens that have been recently characterized as targets of autoantibodies in limbic encephalitis include glutamate receptors of types NMDA (Figure 1) and AMPA, GABAB receptors, LGI1, CASPR2, and DPPX.
Anti-NMDA receptor encephalitis
Anti-NMDA receptor encephalitis is an inflammatory encephalopathic autoimmune disease, which is characterized by highly specific autoantibodies against glutamate receptors of type NMDA (N-methyl-D-aspartate). The disease was first described in 2007 and is currently still widely underdiagnosed. It frequently affects young women with ovarian teratoma, but is also observed in older female patients, in women without tumours, in men and in children. Paraneoplastic cases represent 9-55% of the total, depending on age, gender and ethnicity. Patients usually present with symptoms such as memory loss, disorientation, confusion, paranoid thoughts, visual or auditory hallucinations, dyskinesias, decrease in consciousness, lethargy, seizures and autonomic instability.
Detection of anti-glutamate receptor (type NMDA) autoantibodies constitutes a key criterion for the diagnosis of anti-NMDA receptor encephalitis. Their analysis is particularly important for differential diagnosis in patients with encephalitis of unknown origin, i.e. non-infectious etiology, and in young women with de novo epilepsy. When a positive serological result is obtained, a comprehensive teratoma investigation should be undertaken.
Anti-AMPAR, GABABR and LGI1/CASPR2/DPPX encephalitides
Limbic encephalitides triggered by autoantibodies against glutamate receptors of type AMPA, GABAB receptors, LGI1, CASPR2 or DPPX occur less frequently than anti-NMDA receptor encephalitis. The different disease subtypes manifest with varying symptom complexes, encompassing memory deficits, seizures, confusion, disorientation, neuromyotonia, agitation, behavioural problems, hallucinations, paranoia, hyponatremia, myoclonus, dysautonomia and sleep or consciousness disturbances. Tumours are found with differing frequencies, more commonly in patients exhibiting anti-AMPA (70%) or anti-GABAB receptor (60%) antibodies than in individuals with anti-LGI1 (<20%) or anti-CASPR2 (<20%) positivity. Limbic encephalitis associated with anti-glutamate receptor (type AMPA) antibodies occurs predominantly in women, while the anti-GABAB receptor and LGI1/CASPR2 subtypes are found more frequently in men. Anti-CASPR2 antibodies are also observed in acquired neuromyotonia and Morvan’s syndrome.
The inflammatory autoimmune disease neuromyelitis optica (NMO) is a rare form of the group of acquired demyelinating diseases of the central nervous system. It is characterized by degradation of the insulating sheath of at least one optical nerve and at the same time or a few months later the spinal cord. Symptoms encompass acute visual disorders including blindness, impaired mobility and loss of bladder and bowel control. Without adequate therapy, half of patients become blind in one or both eyes or cannot walk without supports within five years.
Highly specific autoantibodies are found frequently in NMO. The antibodies were first described as NMO-IgG. The protein aquaporin-4 (AQP-4) was later identified as the target antigen. The determination of anti-AQP-4 antibodies is particularly useful for serologically differentiating NMO from classic multiple sclerosis.
Autoantibody detection methods
According to the European Network for Paraneoplastic Neurological Diseases (PNS Euronetwork), antibodies in PNS should always be detected using two unrelated laboratory methods, for example IIFT and immunoblot. The tissue substrates cerebellum, hippocampus, nerve and intestine are used in IIFT for detection of PNS antibodies. Characteristic immunofluorescence patterns indicate the presence of particular autoantibodies (Figure 2). For example, anti-Hu antibodies show granular fluorescence of the neuronal nuclei on cerebellum and hippocampus. With anti-Yo antibodies the Purkinje cells of the cerebellum and to some extent the hippocampus fluoresce. Anti-DNER antibodies can be detected monospecifically using a recombinant-cell substrate of transfected human cells.
The autoantibody specificities obtained in IIFT can be confirmed using line blots containing purified, characterized recombinant antigens. Since the antigens are located at defined positions on the membrane, these blots are extremely easy to evaluate. One of the most extensive panels of neuronal antigens is provided by a new EUROLINE Profile (Figure 3) comprising the twelve antigens: amphiphysin, CV2, Ma2/Ta, Ri, Yo, Hu, recoverin, SOX1, titin, Zic4, GAD65 and Tr (DNER). With this profile, a comprehensive confirmation of anti-neuronal antibodies can be achieved in one simple test.
Autoantibodies against neuronal surface antigens in limbic encephalitis can be detected monospecifically using recombinant-cell IIFT substrates, which consist of transfected human cells expressing defined, well-characterized whole target antigens or the immunoreactive subunits thereof. These tests offer high sensitivity and are easy to evaluate. A further advantage of recombinant-cell IIFT systems is that they can be developed in a short time, often in only a few months. Thus, newly identified autoantibody parameters can progress rapidly from the research laboratory into routine diagnostics. Recombinant-cell substrates are available for the detection of autoantibodies against, for example, glutamate receptors of types NMDA (Figure 4) and AMPA, GABAB receptors, LGI1, CASPR2, DPPX and AQP-4. The efficacy of recombinant-cell assays has been confirmed in various clinical studies. For example, the anti-glutamate receptor (type NMDA) IIFT demonstrated 100% clinical sensitivity and specificity for anti-NMDA receptor encephalitis, while the anti-AQP-4 IIFT yielded a sensitivity of 80% for NMO with a specificity of 100%.
Multiparametric IIFT screening
Since autoimmune neurological syndromes often present with overlapping symptom complexes making them difficult to diagnose, the autoantibody screening should be as wide ranging as possible (1). With IIFT BIOCHIP Mosaics (Figure 5), different tissue and recombinant-cell substrates can be analysed simultaneously. Miniature sections of the substrates are positioned next to each other in each test field of a microscope slide and incubated in parallel with one patient sample. The BIOCHIP slides are incubated using the established Titerplane technique, which provides standardized, parallel incubation of multiple samples under identical conditions. The procedure is thus fast and reliable and is suitable for use in any laboratory familiar with immunofluorescence. Automation options are available to further boost the efficiency and convenience of the analyses.
The importance of multiparametric screening is highlighted by studies on sera sent to a routine clinical immunology laboratory for analysis of anti-neuronal autoantibodies. The sera were subjected to multiplex testing regardless of the test order. In many cases antibody specificities other than those expected were detected.
In a study encompassing 2716 samples submitted over a three-month period (2), 4% were found to be positive for anti-neuronal autoantibodies. Of the positive sera, 67% exhibited positivity for autoantibodies against neuronal surface antigens, compared to 35% for classic intracellular onconeural autoantibodies. Thus, neuronal surface antibodies appear to be more common than classic paraneoplastic autoantibodies. In 31% of the findings, the positive reactions were caused by an autoantibody other than that requested for testing. The antibody frequencies in descending order were NMDAR (42%), CASPR2 (12%), LGI1 (11%), Hu (10%), Ri (9%), Yo (8%), CV2 (6%), GABABR (4%), Ma (2%), PCA-2 (1%) and amphiphysin (1%).
In a second study of 16,741 sera submitted over a time period of one year (3), 14% showed at least one anti-neural autoantibody. From the positive sera that had been submitted for only one monospecific antibody test, 56% contained the requested antibody, while 49% showed a different relevant antibody (5.2% as secondary finding). A wide range of anti-neural antibody specificities was detected. For cell-surface autoantibodies the most common were NMDAR (26%), AQP4 (14%), LGI1 (7%), CASPR2 (7%), GABABR (2%), AMPAR, DPPX, GlyR, MOG, other (1% each), while intracellular autoantibodies were mainly directed against Hu (11%), Yo (10%), GAD (8%), amphiphysin, Ri (2% each), CV2, Ma/Ta, recoverin, Sox1, titin, other (1% each).
Autoantibody testing is an important and increasingly relevant tool for neurologists thanks to the ever expanding portfolio of neuronal autoantibodies. More cases of autoimmune neurological disease are now diagnosed at an early stage, boosting the chances of a favourable outcome for the patients. Of particularly importance is the use of autoantibody parameters as a harbinger of tumour disease. Positivity in neuronal autoantibody tests helps to direct diagnostic efforts towards searching for a neoplasm. In patients with previous cancer, the presence of autoantibodies may signal a return of the malignancy. Patients with non-paraneoplastic autoimmune disorders also benefit from early diagnosis with prompt immunotherapy. Multiparametric assays provide the greatest efficacy in diagnostic screening, as they identify not just the expected parameters, but also secondary reactivities. It is anticipated that ongoing research will identify more relevant anti-neuronal autoantibodies in the future, enabling the diagnostic net to be extended still further.
1. Probst et al. Multiple Sclerosis and Related Disorders 2014: 3 (3): 303-320.
2. Wandinger et al. J. Lab. Med. 2012: 35: 329-42.
3. Komorowski et al. 66th AAN Annual Meeting (Philadelphia, USA) 2014
Jacqueline Gosink, PhD