Recommendations for standardised processing, scoring and reporting of the histopathology from inflammatory arthritis in mice and rats
| Recommendations | Mean level of agreement (SD) |
| Category 1: Sample selection and orientation | |
| 1) Before starting an animal experiment to test a research hypothesis, a sample size calculation should be performed by defining the primary outcome measure, the anticipated effect size, the SD, the power and significance level. | 9,2 (2,3) |
| 2) To maximise standardisation in the evaluation of histopathology of systemic inflammatory arthritis models, hind paws rather than front paws are recommended for analysis. | 9,0 (2,1) |
| 3) Either sagittal or transverse sections can be used for the evaluation of tarsal and/or ankle joints, as long as a standardised orientation is applied. | 9,1 (2,2) |
| 4) To guarantee optimal morphology, paraffin-embedded joint sections rather than cryo-sections should be used for standardised evaluation of histopathology. | 9,7 (0,8) |
| Category 2: Sample preparation, decalcification and staining procedures | |
| 5) Fixation of isolated paws should be performed in 4%–10% formalin for at least 6 hours at room temperature for mice or overnight at 4°C for rats. | 9,6 (1,3) |
| 6) Decalcification should be done in 14% EDTA solution or in 5% formic acid, and compatibility of decalcification agents should be carefully adjusted to planned staining procedures. | 9,7 (0,6) |
| 7) Conventional histological stainings such as H&E, tartrate-resistant acid phosphatase (TRAP), safranin O (SafO) or toluidine blue (TB) staining are recommended for accurate histological analysis of the various joint pathology features. | 9,6 (0,7) |
| 8) Our recommended staining protocols can be used as basic guidelines and will increase standardisation. | 9,8 (0,5) |
| Category 3: General points to consider for scoring histopathology of inflammatory arthritis | |
| 9) For accurate histological scoring, the distinct histopathological features like synovial inflammation, bone erosion, cartilage destruction, proteoglycan depletion and optionally new bone formation should be evaluated as separate parameters. | 9,7 (0,6) |
| 10) Histological scoring of the hind paws should be evaluated in standardised cutting planes and depths for each specimen, and should cover at least three articular joints of ankle/tarsal bones in sagittal sections or at least six tarsal joints in transversal sections. | 9,8 (0,6) |
| 11) Histopathological analysis should be evaluated in at least two (non-serial) sections, simultaneously stained and obtained scores should be subsequently averaged to result in a single data point per animal. | 9,4 (1,3) |
| 12) Histopathological analysis should preferentially be based on the consensus of two independent observers. | 9,1 (1,4) |
| 13) Analysis should be performed in a blinded manner and can be performed using either a semiquantitative scoring system or a quantitative analysis with appropriate software. | 9,9 (0,5) |
| 14) For standardised semiquantitative assessment of the distinct parameters, joint pathology scores should range from 0 (healthy) to 3 (severe) with in-between grading scores of 0.25–0.5 depending on the level of expertise. | 9,4 (1,3) |
| 15) For standardised quantitative analysis of the distinct parameters, joint pathology should be expressed as area (in mm2 of total region of interest) in the case of synovial inflammation, bone erosion, total cartilage and new bone formation, in percentage (% destained cartilage per total cartilage) or as cell counts (in number of positive cells of total region of interest). | 9,4 (1,4) |
| Category 4: Recommendations for evaluating synovial inflammation | |
| 16) Evaluation of synovial inflammation should be performed in H&E-stained sections with 25× magnification for overview purposes and subsequent 50–100× magnification for detailed scoring. | 9,5 (1,1) |
| 17) The degree of synovial inflammation is recommended to be scored either as semiquantitative or quantitative readout parameter as described under 14) and 15). | 9,8 (0,6) |
| 18) A universal, semiquantitative scoring system for synovial inflammation is proposed as: 0, healthy, one to two cell layers of synovial membrane, no inflammatory infiltrates; 1, three to five cell-layered synovial membrane, mild cellular infiltrate into the synovium and exudate in the joint cavity with low cell density; 2, multilayered synovial membranes, enhanced cellular infiltrates and increased cell density throughout the joints; 3, maximally expanded inflammation filling all joint cavities, hyperplastic synovial tissue with high cell density. | 9,6 (0,8) |
| Category 5: Recommendations for evaluating bone erosion | |
| 19) Evaluation of bone erosion should be performed in H&E or TRAP-stained sections under 25× magnification for overview purposes and subsequent 100× magnification for detailed scoring. | 9,8 (0,6) |
| 20) The degree of bone erosion is recommended to be scored either as semiquantitative or quantitative readout parameter as described under 14) and 15). | 9,4 (1,8) |
| 21) In respect to local varieties of the severity of erosions, semiquantitative analyses of bone erosion should be scored as the average calculated for multiple joint areas within one section. | 9,1 (1,7) |
| 22) A universal semiquantitative scoring system for bone erosion is proposed as: 0, healthy, intact bone surface; 1, small focal bone lesions at the surface of cortical bone; 2, enhanced focal, subchondral bone erosions, partial or complete penetration of cortical bone and small breakthrough of cortical bone to bone marrow cavity possible; 3, massive, enlarged erosions of the bone tissue, extended synovial pannus invasion causing complete breakthrough of the cortical bone to the bone marrow cavity, and loss of bone architecture. | 9,5 (1,0) |
| 23) TRAP staining is recommended for further quantification of osteoclasts (as number per total region of interest), where synovial osteoclasts are defined as TRAP+ multinucleated (more than three nuclei) cells within the inflammatory synovial tissue. | 9,8 (0,6) |
| Category 6: Recommendations for evaluating cartilage erosion and proteoglycan loss | |
| 24) Histological scoring of cartilage damage should consist of two major parameters: (1) loss of proteoglycans from the superficial cartilage layer and (2) cartilage erosion of the superficial and/or the deeper calcified cartilage layer. | 9,8 (0,4) |
| 25) Evaluation of cartilage erosion and proteoglycan loss should be performed in SafO or TB-stained sections under 100–200× magnifications for detailed scoring. | 9,7 (0,6) |
| 26) The degrees of cartilage erosion and proteoglycan loss are recommended to be scored either as semiquantitative or quantitative readout parameter as described under 14) and 15). | 9,8 (0,6) |
| 27) In respect to semiquantitative analyses, the severity of cartilage damage should be scored as the average calculated for multiple joint areas within one section. | 9,4 (1,1) |
| Category 7: Data analysis, statistics and reporting | |
| 28) Semiquantitative or quantitative scoring data should be graphically represented, tested for their Gaussian distribution and statistically evaluated by using appropriate parametric or non-parametric tests. | 9,9 (0,3) |
| 29) Representative images of the obtained joint pathology are recommended to be shown to support histological findings. | 9,9 (0,5) |
| 30) To allow standardisation, reproducibility and comparison between different research groups, we recommend to report a minimal dataset to describe the details of the histological procedures and scoring systems, either in the methods section of the main manuscript or as supplemental material in publications. | 9,6 (1,1) |