Newsletter No. 1/2009

The role of antineutrophil cytoplasmic antibodies in the clinical diagnostics of systemic inflammatory disease has been increasing since their description 27 years ago, and they have become an important criterion in vasculitis testing. The current method for the determination of ANCA is indirect immunofluorescence on human neutrophils and antigen-specific ELISAs. The identification of the specific ANCA is necessary for confirming the diagnosis and to recognize clinical variants. It has been established that proteinase 3 (PR3)-ANCA is diagnostically relevant for Wegener's granulomatosis (WG), whereas Myeloperoxidase (MPO)-ANCA is associated with microscopic polyangitis (MPA) (1).

However, the antigen responsible for ca 45% of P-ANCA and 15% of C-ANCA has not been identified. Another ANCA with reactivity toward the antigen, lysosomal-associated membrane protein 2 (LAMP-2), could be detected in patients with focal necrotizing glomerulonephritis (FNGN). This study indicates that antibodies toward LAMP-2 could be detected in nearly all patients with pauci-immune necrotizing glomerulonephritis (sensitivity 93%), but only in one of 20 WG-patients in the localized disease state. Furthermore, the presence of these antibodies correlates very well with disease activity. (2) The diagnostic relevance of LAMP-2 ANCA for other rheumatic diseases has yet to be evaluated.

In addition to their diagnostic value, LAMP-2 ANCA has also contributed to our understanding of the pathogenesis of autoimmune vasculitis (AAV). Kain et al. (2) demonstrated that rats immunized with human LAMP-2 formed antibodies to the protein and developed pauci-immune necrotizing glomerulonephritis.The autoantibodies in AAV patients recognize an important epitope of LAMP-2 (P41-49) that is identical to the bacterial adhesion FimH, a protein in gram-negative bacteria. These antibodies also cross-react with FimH. Rats that were immunized with FimH developed FNGN and antibodies that recognized rat and human LAMP-2. It was shown for the first time that a bacterial infection with gram-negative bacteria (e.g. E. coli) can preclude the development of FNGN. The authors demonstrated that FimH induces an immune reaction toward LAMP-2, and that this mechanism is important in the development of FNGN (2).

ANCA tests for the diagnosis of vasculitides have been in use for over 20 years, but there is still room for improvement. A strategy must be considered that encompasses accuracy, reproducibility and cost-effectivity. In general indirect immunofluorescent techniques (IFT) or antigen-specific direct ELISAs (PR3 and MPO) are used for routine determination of ANCA. However, the prognostic value of IFT can be increased significantly by combining it with standardized antigen-specific ELISAs (3).
An international research group published joint statement regarding ANCA tests (4). The guidelines formulated in this report suggest that an ELISA should be carried out following a positive IFT test. Many clinical laboratories, both in hospitals and independent, use only commercially available ELISA kits for the determination of ANCA. The number of companies that produce these kits has greatly increased; however, the diagnostic efficiency of these assays is not well documented. Since there is no generally recognized international standard, these kits use arbitrary units with unknown interrelationship to IFT titers. In one published study we compared the sensitivity, specificity and predictive value of commercially available direct ANCA ELISA kits with IFT and our in-house PR3 and MPO capture ELISA using sera from patients with clinically and histologically confirmed WG and MPA. This study should lead to the improvement of daily clinical practice and raise the physician's confidence in the test results. The doctor that orders and interprets an ANCA test must be familiar with each assay used and must also know the difference between the various commercially available test systems. The most important conclusions of this study are

1) The performance of commercially available direct ELISAs varies tremendously and is often different than the IFT ANCA; there are significant differences between the sensitivity, specificity and predictive value of commercially available ELISA kits.

2) Only one of 11 direct ELISA kits for WG was comparable with the sensitivity and specificity of IFT (5).

An additional method for the quantification of PR3-ANCA is the capture ELISA. In a capture ELISA the ELISA plate is coated with a specific monoclonal antibody that immobilizes the antigen. There is much data that points to an advantage over the direct ELISA (6). However, the diagnostic value of the diverse capture ELISAs has not been determined, and there are no standardized controls for the available capture tests. The diagnostic specificity and clinical value of the capture PR3-ANCA ELISA as a screening test or for confirming certain forms of vasculitis has yet to be evaluated.
Recently the use of new antigen substrates has enabled the development of more sensitive PR3-ANCA ELISAs. Initial results show that these ELISAs are very sensitive methods for the detection of PR3-ANCA in patients with AAV. Their performance is superior to that of other ELISA methods, and the results correlate to IFT (7, 8).
Currently, the best means of determining vasculitis-associated ANCA is IFT in combination with PR3- und MPO-ANCA ELISA. The solution to problems regarding ANCA-diagnosis is focusing on the fundamental methods, i.e. correct implementation of IFT and ELISA as well as the cautious use of commercial assays. Standard serum reference material for PR3- und MPO-ANCA are available (IUIS/WHO). Furthermore, training workshops aimed at improving ANCA tests should be conducted.