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Analysis of Antineutrophil Cytoplasm Antibody from 118 730 Patients in Tertiary Hospitals in Jiangxi Province, China

Background The discovery of antineutrophil cytoplasm antibody (ANCA) makes the early diagnosis of primary vasculitis possible, and also has important guiding significance for the diagnosis and treatment of secondary vasculitis. This study aimed to investigate the clinical significance of ANCA. Material/Methods ANCA was detected by indirect immunofluorescence assay (IIF), and anti-myeloperoxidase (MPO) antibody, and anti-proteinase 3 (PR3) antibody were detected by ELISA. The results were analyzed retrospectively. Results Among 118 730 patients, a total of 5853 (4.93%) were positive for ANCA. In the positive cases, 3.98% were male and 6.33% were female, with significant differences (χ2=123.38, P<0.01). For ANCA, the department with the highest positive rate (15.06%) was the Department of Rheumatology, followed by 7.78% in the Department of Dermatology, 6.79% in the Department of Nephrology, and 5.72% in the Department of Traditional Chinese Medicine (TCM). Anti-PR3 and cANCA were highly specific in primary vasculitis (P<0.01). Anti-MPO and pANCA had high specificity for other autoimmune diseases (P<0.01). Conclusions ANCA has important guiding significance for vasculitis-related diseases. Therefore, it is important in the diagnosis and treatment of this disease and has value in clinical practice.


Analysis of Antineutrophil Cytoplasm
Background Antineutrophil cytoplasmic antibodies (ANCA) are autoimmune antibodies against neutrophil cytoplasmic components, and has become a specific serological marker antibody for systemic vasculitis represented by Wegener's granulomatosis (WG) in recent years. ANCA, as an important serological diagnosis of vasculitis, is widely recognized by the medical profession for its usefulness in assessing the remission and recurrence status of diseases.
ANCA is divided into cANCA and pANCA, and the matrix tablets for detection of ANCA include ethanol-immobilized neutrophils, formalin-fixed neutrophils, HEP-2 cells, and monkey liver tissues, the latter 2 are mainly to exclude ANA interference in the detection. The main target antigen of pANCA is anti-myeloperoxidase (MPO). MPO is a highly cationic protein with a molecular weight of 146 000, which can cause continuous ribbon fluorescence in the periphery of neutrophils. The main target antigen of cANCA is anti-protease 3 (PR3), which can cause the whole cytoplasmic region of neutrophils to cover coarse particles.
Primary vasculitis is a not yet clear etiology of a class of small vasculitis, mainly violations of small blood vessels, such as small arteries, arteries, vascular wall necrosis inflammation, cellulose-like necrosis as a pathological feature, is a class of sexually transmitted autoimmune diseases. Primary vasculitis generally refers to microscopic multiple vasculitis (MPA), Wegener's granulomatosis (WG), Churg-Strauss syndrome (CSS), and primary necrosis crescentic glomerulonephritis (NCGN). Clinically, WG, MPA, CSS, NCGN, and other series of diseases are called ANCA-related systemic vasculitis (AASV). AASV is a kind of small vascular wall inflammation and/or fibrinoid necrosis, and is the pathological basis of a group of autoimmune diseases, mainly involving systemic small vessel autoimmune disease, and is the most common adult primary vasculitis. ANCA and its target antigen are widely used in clinical practice, and the incidence of AASV is increasing. In this paper, the results of ANCA testing in 118 730 cases in tertiary care hospitals in Jiangxi Province, China, from January 2014 to December 2016 were retrospectively analyzed.

Material and Methods
Review of the cases  [2]. All specimens were obtained with the patient's informed consent and approved by the Hospital Ethics Committee.

Instruments and reagents
Many types of fluorescence microscopes were used, including EUPOStar Plus, Nikon, and OLYMPUS BX. Microplate readers used included SUNRISE, ELX 808, and Multiscan MK3. Washing machines used included PW-960 automatic, ELX 50, and BioTek automatic. Reagents used were the antineutrophil cytoplasmic IgG test kit, Anti-MPO assay kit, and the anti-PR3 assay kit provided by EUROIMMUN (Germany).

Methods
ANCA fluorescence pattern was determined by IIF method. Reagents were purchased from EUROIMMUN Company (Germany). The reaction areas included HEp-2 cells, primate liver tissue, and formaldehyde-and ethanol-fixed human neutrophils. Samples were diluted with PBST buffer for 1: 10 dilution and tested in strict accordance with the EUROIMMUN 4313 titration plate technique. The above steps were in strict accordance with the reagent manual and the standard operating procedure (SOP) in our laboratory.
Human serum anti-MPO and anti-PR3 antibodies were tested by enzyme-linked immunosorbent assay (ELISA) method. Reagents were from EUROIMMUN Company. The samples were diluted, then the diluted sera, negative control, positive control, and blank control were added to the microwells. The platelets were incubated for 30 min at room temperature. After incubation, plates were washed. Human IgG bound on the plates was detected by horseradish peroxidase-conjugated antibody using tetramethylbenzidine (TMB) as a peroxidase substrate. The reaction was stopped by the addition of 2 M H 2 SO 4 , and the absorbance was read at 450 nm. The above steps were in strict accordance with the reagent manual and the SOP.

Statistical analysis
Statistical analyses were performed using SPSS 19.0 (SPSS for Windows, ver. 19.0). Quantitative variables are presented as mean ±SD. The validity was checked using the rank sum test. The c 2 test was used to compare categorical variables. P<0.05 was considered as statistical significance.

Results
The analysis of total positive rate Among 118 730 cases with ANCA test results, there were 5853 positive cases. There was a significant difference between the positive male and positive female groups (c 2 =123.38, P<0.01) ( Table 1).
The departmental distribution of the 5853 cases positive for ANCA The department with highest positive rate was the Department of Rheumatology, which was consistent with the specificity of ANCA-positive status in vasculitis patients treated in this department (Table 2).
Test results of anti-PR3, anti-MPO, and ANCA antibody in 5853 positive cases Anti-PR3 and cANCA pattern were highly specific in vasculitis. The anti-MPO and pANCA pattern had high specificity for other autoimmune disease ( Table 3).

Comparison of the concentration of anti-PR3 and anti-MPO antibody in autoimmune diseases
In the vasculitis group, the concentration and positive rate of anti-PR3 antibody was highest, and in other autoimmune diseases the concentration and positive rate of anti-MPO antibody was highest, as shown in Table 4.
The positive rate in the different types of diseases positive for ANCA The positive rate of ANCA in vasculitis was higher than that in other autoimmune diseases. The positive rate of Wegener's granuloma was the highest in the vasculitis group (71.60%). The positive rate of systemic lupus erythematosus was the highest (6.13%) in the other autoimmune diseases group ( Table 5).  Table 6.

Organ involvement accompanied by 2 patterns of ANCA antibody
The positive rate of pANCA was higher than that of cANCA in cases of kidney involvement, liver involvement, ear involvement, nasal involvement, muscle involvement, nerve system involvement, and digestive tract involvement. The positive rate of cANCA was more than that of pANCA in cases of eye involvement and joint involvement. See details in Table 7.
Clinical evaluation of anti-PR3, anti-MPO, cANCA, and pANCA Anti-PR3 and cANCA were more sensitive and specific, as shown in Table 8.

Discussion
In 1982, Davies [4] detected an IgG type of antibody in patients with segmental necrotizing glomerulonephritis in serum by the IIF method. The antibody antigen is neutrophil cytoplasmic antigen, so it is called ANCA. Later, Van der Woude [5] confirmed the presence of ANCA in serum of patients with Wegener's granulomatosis and identified a specific serological diagnostic value. The important clinical role of ANCA was recognized.
We retrospectively analyzed the test results of 118 730 cases in tertiary hospitals for ANCA from January 2014 to December 2016 in Jiangxi Province. There were 5853 positive cases (4.93%), which is similar to rates in Chinese and foreign reports. For the positivity of ANCA, the male-to-female ratio was 1: 1.85, which is consistent with the literature [6][7][8][9][10][11].
The department with the highest positive rate (15.06%) of ANCA was the Department of Rheumatology in tertiary care hospitals in Jiangxi Province, which is consistent with the specificity of ANCA in rheumatism. The positive rate in dermatology was up to 7.78%, because a common complication of vasculitis is skin rash. The renal positive rate can be up to 6.79%, which is related to ANCA-associated vasculitis, mainly involved in kidney disease, such as rapid progressive glomerulonephritis and necrotic crescentic nephritis [12][13][14].
Among 2053 cases of vasculitis and 3612 cases of other autoimmune diseases, anti-PR3 and cANCA were highly specific in vasculitis. Anti-MPO and pANCA had high specificity for other autoimmune diseases, which is consistent with the literature [25-31.  4316 has a high degree of specificity. There were 457 cases of other autoimmune diseases with anti-MPO antibody concentration of 74.58±41.10 IU/ml, and there were significant differences from the vasculitis group. MPO antibody was highly specific in other autoimmune diseases. This result is consistent with previous studies [32][33][34][35][36][37][38][39][40][41].
Initially, it was found that ANCA existed in the sera of patients with primary vasculitis. As the study of ANCA became more comprehensive, and with the wider clinical use of ANCA, the value of ANCA in diagnosis and prognosis of diseases is increasingly recognized in the medical field. ANCA can be divided into cANCA and pANCA according to the different patterns of immunofluorescence. Some target antigens and some patterns of ANCA are recognized to have a close relationship with some diseases. In our study, anti-PR3 and cANCA were mainly seen in Wegener's granuloma, while anti-MPO and pANCA were mostly seen in microscopic multiple vasculitis. This result is slightly different from Martínez Téllez [42] because the Chinese and Western ANCA-related target antigen corresponding to the disease is different. ANCA contains a variety of antigenic components: mainly anti-MPO and anti-PR3. Additionally, among 5853 patients positive for ANCA, we did not detect anti-MPO or anti-PR3 antibodies in 1034 patients, indicating that in addition to the 2 common target antigens, there were other antigens present, including lysozyme, cathepsin, elastase, and lactoferrin, which is consistent with the literature [43][44][45][46][47][48][49][50]. Further confirmation of other specific target antigens needs to be confirmed.   Table 6. Positive rate of anti-MPO, anti-PR3 antibody, and 2 patterns of ANCA in different diseases.

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In 5853 cases with positive results for ANCA, 2053 cases were diagnosed with vasculitis. PR3-ANCA in Wegener's granuloma had a significant positive rate of 75.14%, while the positive rate of MPO-ANCA was 54.32% in microscopic multiple vasculitis. It was slight lower compared with the results from Zhang [51], and there is a huge difference between the distribution of patients in different cities and using different detection methods. Eosinophilic granulomatous vasculitis in AAV is rare, and this might be related to the low incidence rate in China, and is in line with the literature [52][53][54][55][56][57][58][59]. In 3612 cases of other autoimmune diseases, clinical diseases were mainly SLE-based (20.19% positive) and RA-based (14.10% positive), and given priority to anti-MPO and pANCA. Ulcerative colitis is a type of autoimmune disease with a high positive rate. The anti-MPO and pANCA had a high specificity, which is basically consistent with previous reports [60,61].
ANCA-associated vasculitis mainly involves the kidneys and liver. The positive rate can be up to 25.35% and 23.87%. In addition, the involvement of joints, muscles, nervous system, and digestive tract suggest multi-organ involvement. The positive rate of pANCA was higher than that of cANCA in cases involving the kidneys, liver, ears, nose, muscles, nerves, and digestive tract. The positive rate of cANCA was higher than that of pANCA in cases of eye involvement and joint involvement, consistent with the literature [62,63]. Organ involvement with ANCA-associated vasculitis has a variety of clinical manifestations, resulting in different diseases. Hence, this characteristic causes various difficulties in clinical diagnosis, which need to be explored in greater detail.
There were some limitations to the present study. The first is the limitations of indirect immunofluorescence interpretation. According to the different staining sites of immunofluorescence, ANCA can be divided into cANCA and pANCA. Each

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hospital has subjective factors when reading data from the fluorescent microscope. The second is the limitations of the target antigen. The 2 major target antigens of ANCA are anti-MPO and anti-PR3 (as studied in this paper). However, there are many ANCA-associated target antigens, and the diseases associated with these target antigens in this study are included in the "other diseases" because of the small clinical base, which is not covered in this article and needs further studies. The last one is the limitations of the study object. The clinical data, which was collected from China, were slightly different from the foreign reports.

Conclusions
ANCA enables early diagnosis and treatment of primary vasculitis. With the development of ANCA detection, and as detection technology improves, the detection rate of ANCA in diseases is increasing and more types of diseases can be detected through this method. Therefore, much more attention should be paid to ANCA in the diagnosis of clinical disease.