Design and Preparation of Diagnostic Slides for Antineutrophil Cytoplasmic Antibodies (ANCA) by Indirect Immunofluorescence Method

One of the most important tests for the detection of antibodies against neutrophil cytoplasm in autoimmune vasculitis diseases is the Antineutrophil Cytoplasmic Antibodies (ANCA)  test. The ANCA test is the first order in the initial diagnosis and screening of patients with suspected autoimmune diseases, including small-vessel vasculitis, as well as the follow-up of patients' treatment, which uses indirect immunofluorescence assay (IFA) to detect the presence of antibodies against antigens in the cytoplasm of neutrophils. In addition to vasculitis, the evaluation of this antibody is used in other autoimmune diseases such as systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), and inflammatory bowel diseases (IBD). To prepare IFA slides for diagnostic purposes, neutrophils extracted from human peripheral blood are used as substrates. The IFA is a common and acceptable method in medical diagnostic laboratories for the detection of these autoantibodies. The first experience in the development of this technique back to the work of Koons and Kaplan, although today, this method has been widely developed in the field of laboratory diagnosis as well as research in basic medical sciences. A distinct advantage of the immunofluorescence assay is its applicability to fresh tissue and fixed cells. This method is based on the specific antigen binding to specific antibodies conjugated with fluorescent dyes and fluorescent light emission. There is two common types of immunofluorescence assay include direct and indirect immunofluorescence used for detection autoantibodies. In the direct immunofluorescence method, specific antibodies are conjugated to fluorescent dyes, and by binding to their specific antigen, the green fluorescent light is recognized under a fluorescent microscope. Although this procedure is faster, it is less often applied in medical diagnostic labs. In the IFA method, a specific antibody against the target antigen (called the primary antibody) binds to the target antigen recognized by a secondary antibody that is against the fixed region of the primary antibody and conjugated with fluorescent dyes, such as fluorescein isothiocyanate (FITC). In the indirect immunofluorescence assay, due to the binding of several secondary antibodies to the primary antibody, the generated fluorescence signals will be amplified, which in turn increases the fluorescence intensity and sensitivity. The aim of this study was to design and prepare slides for antibodies detection against antigens in cytoplasm neutrophils by the indirect immunofluorescence method. Furthermore, the next step by optimizing fluorescent patterns to detect different types of antibodies against neutrophil cytoplasm by indirect immunofluorescence method. Since 1980, the search for antibodies against the neutrophil cytoplasm has been recognized as one of the most effective diagnostic tools for diseases associated with small to medium-sized vasculitis. ANCA in the diagnosis and follow-up of the treatment of vasculitic diseases including granulomatosis with polyangiitis, formerly known as Wegener granulomatosis, microscopic polyangiitis, Eosinophilic granulomatosis with polyangiitis, formerly known as Churg – Strauss syndrome, is used. Most laboratories around the world use IFA for screening and early detection of ANCA.After the ANCA test is positive, the target antigens of the antibodies are evaluated by the ELISA method. The two most common targets of autoantibodies are myeloperoxidase (MPO) and proteinase-3 (PR-3) in patients with vasculitis.However, a clinical association of these vasculitis diseases with other antigens such as elastase, lactoferrin, azurocidin, lysozyme, H-Lamp-2, and other lesser-known antigens has also been observed.Antibodies against neutrophil cytoplasm have also been reported in other small vessel vasculitis diseases such as microscopic polyangiitis (MPA), eosinophilic granulomatosis with polyangiitis (EGPA), and necrotizing crescentic glomerulonephritis. Initially, the non-commercial ELISA method was used to diagnose PR3-ANCA and MPO-ANCA, but inconsistent results between laboratories showed the global need for standardization of this test . In 1998, fifteen laboratory centers around the world evaluated and standardized the standard method for measuring antibodies against neutrophil cytoplasm (ANCA) and its specific antigens, including PR3 and MPO, by indirect immunofluorescence and ELISA in patients with idiopathic systemic vasculitis. . The result of this standardization increased the detection value of ANCA by immunofluorescence and ELISA methods.One of the most important pillars of the commercialization of a product is the supply of raw materials with appropriate and sustainable quality. The first step in producing ANCA slides is the isolation of high purity and standard quality peripheral blood neutrophil cells. Peripheral blood neutrophils are short-lived, highly sensitive cells that cannot be stored for long periods of time, and it is recommended that these cells be isolated from whole blood immediately after blood sampling. To date, several methods have been proposed to isolate neutrophils from peripheral blood. In this study, it was shown that using the dextran method and subsequent separation by centrifugal gradient method with filament is one of the cost-effective methods with high purity to provide the ANCA test substrate.

Using the Ficoll density gradient centrifugation and dextran solution, neutrophils were isolated from the peripheral blood of healthy individuals (without a history of any disease, especially autoimmune diseases in themselves and first-degree relatives). The purity and efficiency of the isolated cells were evaluated by Wright Giemsa staining, the neobar slide, and an automatic counting machine. Isolated neutrophils were fixed on slides using an ethanol fixative. At this stage, by selecting the best protocol, in terms of time and type of neutrophil fixation stage, they were fixed on the slide. In the next step, using positive and negative samples that have already been determined by ANCA with valid commercial kits (IVD certified), the slides containing fixed cells were fluorescent stained and compared with control samples.

The number and purity of neutrophils isolated were evaluated by a Sysmex automatic cell counter. Depending on the peripheral blood neutrophils, the mean(±standard deviation) of the number of isolated neutrophils was 6000(±950) cells per microliter. According to white blood cell differential count, the mean purity of isolated neutrophils was 94.1%. Also, by trypan blue staining, the percentage of viable cells was determined to be more than 85% before fixation. The results of ANCA indirect fluorescent staining on the prepared slides were completely consistent with the results of control samples. 

The results of this study showed that the method used can be applied for the commercialization and production of ANCA slides. In this regard, in addition to optimizing the steps of separation and fixing the neutrophils, it is necessary to evaluate and optimize the stability of the prepared slides during storage and production in order to produce in large numbers.