Clinical data collection
General clinical data from patients with axSpA who underwent SIJ fine needle aspiration biopsy were collected, including age, gender, course of the disease, initial site, and symptoms. Laboratory tests included measurement of human leukocyte antigen B27 (HLA B27), erythrocyte sedimentation rate (ESR), and C-reactive protein (CRP) levels. Radiographs of the pelvis and lumbar spine, and CT/magnetic resonance imaging (MRI) data on the SIJ were also obtained.
Diagnostic criteria
The diagnosis of axSpA was made according to the axSpA classification criteria established by the ASAS, as revised in the New York criteria [9]. Subjects with axSpA who did not exhibit radiological sacroiliitis and did not meet the New York criteria were defined as having non-radiographic axSpA (nr-axSpA) [10].
MRI edema scoring
The degree of bone marrow edema in the SIJ was graded using the Spondyloarthritis Research Consortium of Canada criteria [11]. Oblique coronal plane scanning was adopted by selecting six consecutive synovial areas (sacrum and sacral foramen of the lateral sacrum, not including synovial and ligaments). Each joint was divided into four quadrants that constituted the upper and lower part of the ilium and sacrum at all levels. Any quadrant with bone marrow edema received a score of 1, while 0 represented the absence of edema. If the signals of edema were similar to those of blood vessels, then one point was added to this score. If the range of edema below the bone lamella was greater than 1 cm, then an additional point was added to this score. The highest possible score for a quadrant was 12, with the total possible score being 72.
Fine needle aspiration biopsy
Patients in the prone position underwent a biopsy of the SIJ using an FRANSEEN lung biopsy needle (Cook Company, DFBN - 15 or 16–18-15), guided by CT. The intra-articular tissues were extracted by negative pressure [4, 12].
Autopsy specimens
The pelvis was exposed by lateral cutting of the abdomen. The psoas major muscle was cut and stripped toward its site of attachment and as far laterally as possible, thereby exposing the upper part of the SIJ. A saw was used to mutilate both ends of the SIJ away from the articular surface by approximately 2 cm and then the anatomical ends were cut and immediately immersed in 4% neutral formalin.
Tissue processing
All specimens were fixed with 10% formalin solution, decalcified with 10% EDTA solution, dehydrated by an automatic tissue hydroextractor (type: CJ - 14 d2, Dingyuan Hubei Co., Ltd), embedded in paraffin, and sectioned into 4-μm-thick slices.
Immunohistochemistry analysis
Kit I included antibodies against CD34, CD68, vascular endothelial growth factor (VEGF), caspase-3 (rat monoclonal antibody, Zho Zhongshan Gene Co., Ltd., Beijing), type I collagen (rabbit polyclonal antibody. Zhongshan Gene Co., Ltd., Beijing, China), metalloproteinase-3 (MMP-3; rabbit polyclonal antibody; Abcam Co., Ltd., USA), and tumor necrosis factor-α (TNF-α; rabbit polyclonal antibody; Boster Co., Ltd., Wuhan, China). Immunohistochemistry kit II included a two-step mouse and rabbit ultra-sensitive PV immunohistochemistry ELISA kit with a two-step (polymer auxiliary agent + IgG polymer labeled with horseradish enzyme; Zhongshan Gene Co., Ltd., Beijing, China) SAP immunohistochemistry detection kit (blocked with normal sheep serum, goat anti-mouse/rabbit IgG labeled with biotin and chain mildew-avidin labeled with alkaline phosphatase; Zhongshan Gene Co., Ltd., Beijing, China). Staining reagents included ready-to-use hematoxylin dye solution (Huntz Enterprises Inc., Shanghai, China), eosin (Solarbio Inc., Shanghai, China), and safranin and fast green stain solution kits (IHC World Co., Ltd., USA). All sections were stained with hematoxylin-eosin and safranin O-fast green. Some of sections were stained using the immunohistochemistry reagents described above. Slides were incubated with primary antibody overnight at 4 °C, and with secondary antibody for 15 min at 37 °C. Slides were then visualized with 3,3-diaminobenzidine (DAB) or AP-Red.
Microscopic examination
The various pathologic changes in the biopsy specimens were observed using microscopy. These changes were compared with healthy autopsy specimens.
Definition of pathologic changes in sacroiliitis
Pathologic changes were defined as follows:
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1.
Cartilage degeneration: chondrocyte hyperplasia or hypertrophy, or focal distribution; cartilage matrix depletion (stained with hematoxylin-eosin and safranin O-fast green), or fibrosis and mucoid degeneration.
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2.
Endochondral ossification: bone deposits on remnants of the cartilage matrix.
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3.
Pannus formation: highly vascular granulation tissue formed from the inflamed synovium or subchondral bone marrow.
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4.
Subchondral bone disruption: pannus invasion and destruction of the subchondral bone.
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5.
Osteoclast activation: formation of at least five CD68+ multinuclear osteoclasts in areas of bone resorption at the subchondral bone endplate or at the bone-cartilage interface, which are expressed as the total number per 10 high-power fields (hpf).
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6.
Sequestrum: a fragment of bone that has become necrotic and has separated from the normal bone structure.
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7.
Pathologic new bone formation: at the inflamed bone-cartilage interface, accompanied with granulation tissue, new bone tissue forms and is surrounded by osteoblastic layers.
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8.
Marrow inflammatory cell infiltration: mononuclear cells (CD3+ T cells or CD68+ macrophages) aggregated in the subchondral bone marrow, defined as ≥ 50 per 10 hpf.
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9.
Synovitis: inflammation of the synovium with hyperplasia of the synovial lining cells and hyperplasia of the loose connective tissues and interstitial edema, which can be accompanied by inflammatory cell infiltration.
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10.
Enthesitis: iInflammation of the enthesis, with highly vascular dense connective tissue (ligament) and inflammatory cell infiltration.
The density of the micro-vessels (marked with CD34) and the numbers of CD68+ osteoclasts/macrophages at the conjunctional zone of the cartilage and the bone plate were calculated from five continuous visual fields; the number of microvessels and osteoclasts/macrophages were counted and averaged. If the microvessel density was equal to or greater than 10 per 40 hpf, it was defined as an increase in angiogenesis.
VEGF, MMP3, TNF-α and caspase-3 expression was semiquantitatively evaluated from five visual fields (10 × 20 magnification), collected continuously at the site of the cartilage and the bone plate, and at the sites of bone marrow beneath the bone plate. We studied the biopsy specimens and evaluated the ratio of cells with positive expression in each visual field. A ratio ≤ 5% was scored as 1 point; 6–25% was scored as 2 points; 26–50% was scored as 3 points; 51–75% was scored as 4 points; and > 75% was scored as 5 points. We also employed a quantitative method of assessing expression using Image-Pro Plus to calculate the area of positive expression in each visual field (10 × 20 magnification under the same condition of exposure). The average score for multiple visual fields was then determined.
Statistical analysis
SPSS 17.0 software was used for statistical analysis. The chi-square (χ2) test was applied to analyze the enumeration data. Measurement data are expressed as mean ± standard deviation and the normality of the measurement data was first tested; the t test was applied to normally distributed data and the rank sum test was used for non-normally distributed data. Binary multivariate regression analyses were applied to analyze the factors that influence the progression of SIJ imaging in different pathologic manifestations of cartilage and bone plate. The Mann-Whitney U test was used to analyze the immunohistochemical results. Statistical differences were assumed to be significant when the p value was < 0.05.