The Authors wish to acknowledge Professor Leonardo Punzi for his precious mentorship in the field of synovial fluid analysis and Padova University Hospital for its support.

The present protocol complies with the guidelines of the Ethics committee of Padova University Hospital. SF was collected with patient consent from the knee joints of patients receiving therapeutic arthrocentesis for joint effusion at their initial presentation to the clinic or in response to an arthritic flare. Contraindications to the procedure were: coagulopathy, anticoagulant medications, skin lesions, dermatitis, or cellulitis overlying the joint. All SF samples were deidentified.
1. Synovial fluid preparation
<ol>
	<li>Transfer the SF samples collected during arthrocentesis of swollen joints15 into a tube containing anticoagulant (preferably EDTA; see Table of Materials) and to a second tube containing no additive (Figure 1).</li>
	<li>In suspected cases of infection, aseptically transfer a portion of the SF sample (~ 1 mL) into culture bottles for microbiological testing. 
	NOTE: Infection can be suspected as a result of both clinical features (severe tumor, dolor, calor, functio laesa, and at times fever) and macroscopic appearance of the SF (turbidity, color). In this case, the physician prescribes a microbiological analysis, which is performed by a microbiology laboratory.</li>
	<li>Carry out SF analysis (steps 2-5) as soon as the sample is collected.</li>
</ol>
2. Macroscopic examination
NOTE: Macroscopic evaluation of SF samples consists of subjective, qualitative, and semiquantitative assessments. There are no reference standard scales, and no control samples are used.
<ol>
	<li>Annotate the SF volume expressed in milliliters. Define the color of the sample, which can range from pale to dark yellow. Record the presence of blood.</li>
	<li>Define the degree of clarity by observing the sample in the backlight and determining the ease of reading a printed page through the fluid tube (Figure 2A).</li>
	<li>Assess the viscosity by dripping the sample from a disposable pipette. A long string or drop formation indicates good or poor viscosity, respectively (Figure 2B).</li>
	<li>Assess the presence of particulate materials, including tissue fragments or fibrin, by observing the sample through the tube and in the backlight.</li>
</ol>
3. Microscopic examination
NOTE: The SF microscopic analysis consists of cytological examination, including total and differential white blood cell (WBC) counts and the search for crystals.
<ol>
	<li>Determine the total cell count following the steps below.
	<ol>
		<li>Determine the total number of leukocytes in the SF collected in the EDTA tube using a hemocytometer (see Table of Materials).</li>
		<li>Draw 0.5 &#181;L of the SF from the EDTA tube using a Malassez-Potain pipette (mark 0.5; see Table of Materials) (Supplementary Figure 1). With the same pipette, draw 9.5 &#181;L of 0.1% methylene blue solution (mark 11). The final dilution is 20-fold.</li>
		<li>Mix the pipette by gentle inversion, discard the first drops, and pour the synovial fluid + methylene blue solution into the counting chamber.</li>
		<li>Count the cells in two consecutive squares out of nine, and multiply the obtained number by 100 (simplified formula; Supplementary Figure 2). Express WBCs as the number of leukocytes per cubic millimeter. 
		NOTE: Simplified formula: Leukocytes (N/mm3) = the number of counted cells in two consecutive squares x 100.</li>
		<li>Classify the SF according to the total number of WBCs following previously published reports16,17 (Table 1).</li>
	</ol>
	</li>
	<li>Perform differential cell count.
	<ol>
		<li>Determine the percentage of polymorphonuclear (PMN) cells, monocytes, and lymphocytes using supravital or traditional staining13,18.</li>
		<li>For supravital staining, place one small drop of SF in the center of a ready-to-use slide pre-coated with cresyl violet and methylene blue (Supplementary Figure 3; see Table of Materials).</li>
		<li>Cover with a coverslip, and place a drop of microscope immersion oil.</li>
		<li>Prepare an air-dried SF smear for traditional staining (less suitable for routine analysis). Put a small drop of SF at the end of a clean slide. Using a second slide or a coverslip, spread it along the slide with a forward movement. Let the smear air-dry.</li>
		<li>Stain the sample according to a standard May-Gr&#252;nwald-Giemsa procedure18.</li>
		<li>Examine the slide under the oil immersion objective (1,000x).</li>
		<li>Count 100 consecutive cells and distinguish between polymorphonuclear cells, monocytes, and lymphocytes (Figure 3, Supplementary Figure 4).</li>
		<li>Express the total number in each category as a percentage. 
		&#8203;NOTE: To obtain accurate results, cytological examinations must be performed as soon as the SF is collected or within 24-48 h of arthrocentesis when properly stored (4 &#176;C).</li>
	</ol>
	</li>
</ol>
4. Crystal detection
<ol>
	<li>Determine the presence of pathological crystals using a polarized light microscope equipped with an additional polarizing filter and a red compensator19 (see Table of Materials).</li>
	<li>Perform slide preparation for crystal detection.
	<ol>
		<li>Carefully clean a glass slide with optical paper or soft paper imbibed with ethanol. Apply a small drop of SF on the glass slide.</li>
		<li>Cover with a coverslip and place the slide under the microscope.</li>
	</ol>
	</li>
	<li>Identify the crystals.
	<ol>
		<li>Focus the slide under the microscope using a low-power magnification objective. Carefully examine the slide under a bright field with a 40x objective. Look inside and outside the cells.</li>
		<li>Identify the crystals according to their size and shape (e.g., rhomboidal, needle-shaped, rod-shaped).</li>
		<li>Insert the polarizing filter between the light source and the specimen. Rotate the polarizer until its optical axis is perpendicular to the analyzer (placed above the objectives) to create a dark background.</li>
		<li>Once a birefringent crystal has been identified, insert the compensator between the polarizer and the specimen and move it parallel or perpendicular to the optical axis of the crystal.</li>
		<li>Observe the color revealed by the crystal (Table 2). 
		NOTE: MSU crystals appear needle-shaped, brightly birefringent in the dark field, and display a yellow/orange color when their axis is parallel to the compensator. Conversely, they are blue when positioned perpendicular (negative sign) (Table 2; Figure 4). CPP crystals are rod- or rhomboid-like crystals, weakly birefringent under polarized light, and display a blue or yellow color when they are aligned parallel or perpendicular to the axis of the compensator (positive sign), respectively (Table 2; Figure 5).</li>
	</ol>
	</li>
</ol>
5. Alizarin red staining
<ol>
	<li>Prepare a 2% solution of alizarin red S (pH 4.1-4.3), filter the solution through a 0.22 &#181;m filter, and store it away from light.
	<ol>
		<li>For preparing the slide, mix a drop of SF with the same volume of alizarin red solution (1:1) on a clean slide. Cover with a coverslip and examine at 400x magnification.</li>
		<li>Identify the crystals under the microscope. 
		NOTE: The test is positive when bright, dark red precipitates are visible under transmitted light, with a round shape and concentric layers. Under polarized light, they appear strongly birefringent20&#160;(Figure 6).</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Cui, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global,+regional+prevalence,+incidence+and+risk+factors+of+knee+osteoarthritis+in+population-based+studies.">Global, regional prevalence, incidence and risk factors of knee osteoarthritis in population-based studies.</a> EClinicalMedicine. 29, 100587 (2020).</li><li>Hunter, D. J., Bierma-Zeinstra, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Osteoarthritis.">Osteoarthritis.</a> Lancet. 393 (10182), 1745-1759 (2019).</li><li>Mandell, B. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Synovial Fluid Analysis and the Evaluation of Patients with Arthritis. , (2022).</li><li>Schmerling, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Guidelines+for+the+initial+evaluation+of+the+adult+patient+with+acute+musculoskeletal+symptoms.">Guidelines for the initial evaluation of the adult patient with acute musculoskeletal symptoms.</a> Arthritis &amp; Rheumatism. 39 (1), 1-8 (1996).</li><li>Coakley, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RCGP+and+BSAC+guidelines+for+management+of+the+hot+swollen+joint+in+adults.">RCGP and BSAC guidelines for management of the hot swollen joint in adults.</a> Rheumatology. 45 (8), 1039-1041 (2006).</li><li>Frallonardo, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Basic+calcium+phosphate+and+pyrophosphate+crystals+in+early+and+late+osteoarthritis:+relationship+with+clinical+indices+and+inflammation.">Basic calcium phosphate and pyrophosphate crystals in early and late osteoarthritis: relationship with clinical indices and inflammation.</a> Clinical Rheumatology. 37 (10), 2847-2853 (2018).</li><li>Nalbant, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synovial+fluid+features+and+their+relations+to+osteoarthritis+severity:+new+findings+from+sequential+studies.">Synovial fluid features and their relations to osteoarthritis severity: new findings from sequential studies.</a> Osteoarthritis Cartilage. 11 (1), 50-54 (2003).</li><li>Fuerst, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcification+of+articular+cartilage+in+human+osteoarthritis.">Calcification of articular cartilage in human osteoarthritis.</a> Arthritis &amp; Rheumatism. 60 (9), 2694-2703 (2009).</li><li>Campillo-Gimenez, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inflammatory+potential+of+four+different+phases+of+calcium+pyrophosphate+relies+on+NF-&#954;B+activation+and+MAPK+pathways.">Inflammatory potential of four different phases of calcium pyrophosphate relies on NF-&#954;B activation and MAPK pathways.</a> Frontiers in Immunology. 9, 2248 (2018).</li><li>Paz&#225;r, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Basic+calcium+phosphate+crystals+induce+monocyte/macrophage+IL-1&#946;+secretion+through+the+NLRP3+inflammasome+in+vitro.">Basic calcium phosphate crystals induce monocyte/macrophage IL-1&#946; secretion through the NLRP3 inflammasome in vitro.</a> The Journal of Immunology. 186 (4), 2495-2502 (2011).</li><li>Mart&#237;n, M. J. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Automated+cell+count+in+body+fluids:+a+review.">Automated cell count in body fluids: a review.</a> Advances in Laboratory Medicine. 2, 149-161 (2021).</li><li>Seghezzi, , et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+of+cellular+analysis+of+synovial+fluids+by+optical+microscopy+and+automated+count+using+the+Sysmex+XN+body+fluid+mode.">Optimization of cellular analysis of synovial fluids by optical microscopy and automated count using the Sysmex XN body fluid mode.</a> Clinica Chimica Acta. 462, 41-48 (2016).</li><li>Reginato, A. J., Maldonado, I., Reginato, A. M., Falasca, G. F., O'Connor, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Supravital+staining+of+synovial+fluid+with+Testsimplets.">Supravital staining of synovial fluid with Testsimplets.</a> Diagnostic Cytopathology. 8 (2), 147-152 (1992).</li><li>Lumbreras, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+for+crystals+in+synovial+fluid:+training+of+the+analysts+results+in+high+consistency.">Analysis for crystals in synovial fluid: training of the analysts results in high consistency.</a> Annals of the Rheumatic Diseases. 64 (4), 612-615 (2005).</li><li>Tieng, A., Franchin, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Knee+arthrocentesis+in+adults.">Knee arthrocentesis in adults.</a> Journal of Visualized Experiments. , e63135 (2022).</li><li>Punzi, L., Oliviero, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arthrocentesis+and+synovial+fluid+analysis+in+clinical+practice:+value+of+sonography+in+difficult+cases.">Arthrocentesis and synovial fluid analysis in clinical practice: value of sonography in difficult cases.</a> Annals of the New York Academy of Sciences. 1154 (1), 152-158 (2009).</li><li>Schumacher, H. R., Reginato, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Atlas of Synovial Fluid Analysis and Crystal Identification. , (1991).</li><li>Mopin, A., Driss, V., Brinster, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detailed+protocol+for+characterizing+the+murine+C1498+cell+line+and+its+associated+leukemia+mouse+model.">Detailed protocol for characterizing the murine C1498 cell line and its associated leukemia mouse model.</a> Journal of Visualized Experiments. (116), e54270 (2016).</li><li>Oliviero, F., Punzi, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Basics+of+Polarized+Light+Microscopy.">Basics of Polarized Light Microscopy.</a> Synovial Fluid Analysis and the Evaluation of Patients with Arthritis. , 79-90 (2022).</li><li>Paul, H., Reginato, A. J., Schumacher, H. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alizarin+red+S+staining+as+a+screening+test+to+detect+calcium+compounds+in+synovial+fluid.">Alizarin red S staining as a screening test to detect calcium compounds in synovial fluid.</a> Arthritis &amp; Rheumatism. 26 (2), 191-200 (1983).</li><li>Favero, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synovial+fluid+fetuin-A+levels+in+patients+affected+by+osteoarthritis+with+or+without+evidence+of+calcium+crystals.">Synovial fluid fetuin-A levels in patients affected by osteoarthritis with or without evidence of calcium crystals.</a> Rheumatology. 58 (4), 729-730 (2019).</li><li>Frallonardo, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+calcium+crystals+in+knee+osteoarthritis+synovial+fluid:+a+comparison+between+polarized+light+and+scanning+electron+microscopy.">Detection of calcium crystals in knee osteoarthritis synovial fluid: a comparison between polarized light and scanning electron microscopy.</a> Journal of Clinical Rheumatology. 22 (7), 369-371 (2016).</li><li>Peffers, M. J., Smagul, A., Anderson, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteomic+analysis+of+synovial+fluid:+current+and+potential+uses+to+improve+clinical+outcomes.">Proteomic analysis of synovial fluid: current and potential uses to improve clinical outcomes.</a> Expert Review of Proteomics. 16 (4), 287-302 (2019).</li></ol>

All authors disclose no conflicts of interest.

In OA, SF analysis helps define disease characteristics through simple steps: total and differential leukocyte counts and searching for crystals, including CPP and BCP. Furthermore, the detection of MSU crystals may highlight important comorbidities.
Despite low costs and simple execution, the sensibility of the tests and reliability of the results may be affected due to inexperienced analysts-mainly as it pertains to crystal identification. Training and experience of the analyst are crucial i...

Osteoarthritis (OA) is a complex and multifactorial chronic joint disease, with an estimated pooled global prevalence of 16% in subjects aged 15 and over, and 23% in subjects aged 40 and over1. The incidence of OA is expected to increase due to an aging population and an increase in risk factors, such as obesity and metabolic syndrome2.
Among the major issues associated with OA are the difficulty in diagnosing the disease in its early stages and currently available treatments limited to pain management and symptomatic slow-acting drugs (SYSADOAs) such as glycosaminoglycans. The diagnosis of OA is based on clinical symptoms and imaging findings. However, the lack of correlation between the two assessments can cause years of delay in OA diagnosis and treatment initiation2. OA is characterized by degenerative processes and low-grade inflammation, which can be easily investigated via synovial fluid (SF) analysis. SF is a viscous plasmatic dialysate rich in hyaluronic acid, which lubricates the joint space, provides nutrients and oxygen to cartilage, and removes metabolic waste. SF also acts as a shock absorber, thus protecting the joints during stress and strain3.
SF analysis is a simple and reliable method that must always be performed during the initial evaluation of patients with musculoskeletal symptoms and joint effusions3. Recommendations from the American College of Rheumatology, the British Society for Rheumatology, and others include SF analysis among the diagnostic testing for rheumatic diseases that must be undertaken mainly in evaluating acute monoarthritis4,5. Total and differential leucocyte counts obtained from SF analysis provide a snapshot of the pathological process occurring in the joint, thus classifying the degree of inflammation. The identification of pathogenic crystals, such as monosodium urate (MSU) and calcium pyrophosphate (CPP) crystals, under polarized light, is vital in the diagnosis and treatment of crystal arthritis (e.g., gout and pseudogout). Furthermore, the presence of microorganisms suggests a diagnosis of septic arthritis.
Calcium crystals are frequent in samples collected from patients with OA6. Basic calcium phosphate (BCP) crystals and CPP have been reported in approximately 22% and 23% of SF samples, respectively, from patients with OA6. Although their role remains unclear, these crystals have been associated with more severe forms of OA7 and are considered an epiphenomenon of the pathological process itself. Calcium crystals have been detected in 100% of tissue samples from OA patients undergoing knee replacement8. Furthermore, it has been hypothesized that calcium crystals may be involved in the pathogenesis of OA owing to their inflammatory effects, demonstrated by several studies9 and mediated, at least in part, by the NLRP3 inflammasome10.
More recently, calcium crystals have been described in both early and late stages6 of OA, indicating that they may play a vital role in diagnosing different clinical subsets of OA and pharmacological treatment.
The overall goal of SF analysis is twofold: to determine the inflammatory degree of SF and to diagnose crystal or septic arthritis by identifying specific crystals or microorganisms. It is a particularly useful, simple, and reliable tool in diagnosing OA, owing to the typical non-inflammatory pattern with a very low percentage or absence of neutrophils.
Advantages over alternative techniques 
SF analysis consists of simple procedures that include total and differential leucocyte counts and crystal search. Manual cell counting performed by expert laboratory technicians3,11 remains the gold standard for the cytological analysis of synovial and other body fluids. However, due to the time-consuming limitation and the inter- and intra-observer variability of this method, automated cell counters have gradually replaced manual counting in large routine clinical laboratories where blood and urine analyzers have been adapted to enable SF analysis11,12. Nevertheless, manual counting presents some advantages in specific settings: (1) in the ambulatory, to obtain a total and differential WBC value on time; (2) to identify cell types such as cytophagocytic mononuclear (Reiter) cells or non-hematopoietic cells such as synoviocytes, that automated counters cannot recognize; (3) when the sample is too small to be handled by the instrument; (4) to create local laboratory registries that are readily accessible for research purposes. Another advantage of manual counting is seen insupravital staining, a method that allows the differentiation of cells very quickly and immediately after glass slide preparation. By contrast, traditional stainings, such as the Wright and the May-Gr&#252;nwald-Giemsa procedures, require air-dried SF smears and time to stain the cells13. Although not suitable for time-sensitive routine analyses, these staining methods reveal more detailed cell populations in the sample, including erythrocytes, basophils, eosinophils, polymorphonuclear leukocytes, lymphocytes, and platelets.
Finally, routine SF analysis for crystals is performed using a simple technique based on polarized light microscopy, which provides fast results14. Alternative methods, such as scanning and electron microscopy, yield more accurate and sensitive results, but their use in everyday clinical practice is not feasible due to high costs and time-consuming sample preparation and analysis.

<table><tbody><tr><td>Alizarin red S</td><td>Merck</td><td>A5533</td><td>For BCP crystal search</td></tr><tr><td>Burker chamber</td><td>Merck</td><td>BR718905</td><td>For total white blood cell count</td></tr><tr><td>Cover glasses</td><td>Merck</td><td>C7931</td><td>For microscopic examination&amp;nbsp;</td></tr><tr><td>EDTA tubes</td><td>BD</td><td>368861</td><td>For SF collection&amp;nbsp;</td></tr><tr><td>Glass slides&amp;nbsp;</td><td>Merck</td><td>S8902</td><td>For crystal search</td></tr><tr><td>Lambda filter (compensator)</td><td>Any&amp;nbsp;</td><td>Refer to microscope company</td><td>For crystal identification&amp;nbsp;</td></tr><tr><td>Malassez-Potain pipette</td><td>Artiglass</td><td>54830000</td><td>For dilution of synovial fluid</td></tr><tr><td>Methylene blue solution</td><td>Merck</td><td>3978</td><td>For total white blood cell count</td></tr><tr><td>Polarized microscope&amp;nbsp;</td><td>Leica, Nikon, others</td><td>Depending on the model and company</td><td>For complete synovial fluid analysis</td></tr><tr><td>Polarizing lens</td><td>Any&amp;nbsp;</td><td>Refer to microscope company</td><td>For crystal identification&amp;nbsp;</td></tr><tr><td>Testsimplet&amp;nbsp;</td><td>Waldeck</td><td>14386</td><td>Supravital staining for cell differentiation</td></tr></tbody></table>

synovial fluid analysis to identify osteoarthritis

Synovial fluid (SF) analysis is important in diagnosing osteoarthritis (OA). Macroscopic and microscopic features, including total and differential white blood cell (WBC) count, help define the non-inflammatory nature of SF, which is a hallmark of OA. In patients with OA, WBC in SF samples usually does not exceed 2000 cells per microliter, and the percentage of inflammatory cells, such as neutrophils, is very low or absent. Calcium crystals are frequent in SF collected from OA patients. Although their role in the pathogenesis of OA remains unclear, they have been associated with a mild inflammatory process and a more severe disease progression. Recently, calcium crystals have been described in both the early and late stages of OA, indicating that they may play a vital role in diagnosing different clinical subsets of OA and pharmacological treatment. The overall goal of SF analysis in OA is two-fold: to ascertain the non-inflammatory degree of SF and to highlight the presence of calcium crystals.

Large joints affected by OA are often swollen and produce significant amounts of SF, which are drained via arthrocentesis15. The macroscopic characteristics of SF evaluated immediately after arthrocentesis essentially include the quantity, color, clarity, and viscosity17. Despite their low specificity, they provide preliminary data on the degree of inflammation. The color depends on SF cellularity and the degree of fragmentation of extracellular matrix macromolecul...

Watch this Scientific Journal Video about Synovial Fluid Analysis to Identify Osteoarthritis at JoVE.com

Synovial Fluid Analysis to Identify Osteoarthritis

Synovial fluid analysis under transmitted, polarized, and compensated light microscopy is used to evaluate the inflammatory or non-inflammatory nature of a sample through simple steps. It is particularly useful in osteoarthritis to detect calcium crystals and identify a more severe subset of osteoarthritis.

Synovial fluid (SF) analysis is important in diagnosing osteoarthritis (OA). Macroscopic and microscopic features, including total and differential white ...

synovial-fluid-analysis-to-identify-osteoarthritis

Research

JoVE Journal

Medicine

4.2K Views.  University of Padova. Synoviale vloeistofanalyse kan de mate van ontsteking en de aanwezigheid van pathogene kristallen onthullen tijdens de eerste evaluatie van patiënten met musculoskeletale symptomen en gewrichtsuitvloeiingen. Het belangrijkste voordeel van deze techniek is de eenvoud en betrouwbaarheid. In een paar stappen kan de clinicus belangrijke diagnostische informatie over de ziekte van de patiënt verkrijgen.De identificatie van specifieke kristallen is van vitaal belang voor diagnose en behand...

Video: Synovial Fluid Analysis to Identify Osteoarthritis

A 3D System for Culturing Human Articular Chondrocytes in Synovial Fluid

Cartilage destruction is a central pathological feature of osteoarthritis, a leading cause of disability in the US. Cartilage in the adult does not regenerate very efficiently in vivo; and as a result, osteoarthritis leads to irreversible cartilage loss and is accompanied by chronic pain and immobility 1,2. Cartilage tissue engineering offers promising potential to regenerate and restore tissue function. This technology typically involves seeding chondrocytes into natural or synthetic scaffolds and culturing the resulting 3D construct in a balanced medium over a period of time with a goal of engineering a biochemically and biomechanically mature tissue that can be transplanted into a defect site in vivo 3-6. Achieving an optimal condition for chondrocyte growth and matrix deposition is essential for the success of cartilage tissue engineering. 
 In the native joint cavity, cartilage at the articular surface of the bone is bathed in synovial fluid. This clear and viscous fluid provides nutrients to the avascular articular cartilage and contains growth factors, cytokines and enzymes that are important for chondrocyte metabolism 7,8. Furthermore, synovial fluid facilitates low-friction movement between cartilaginous surfaces mainly through secreting two key components, hyaluronan and lubricin 9 10. In contrast, tissue engineered cartilage is most often cultured in artificial media. While these media are likely able to provide more defined conditions for studying chondrocyte metabolism, synovial fluid most accurately reflects the natural environment of which articular chondrocytes reside in.
 Indeed, synovial fluid has the advantage of being easy to obtain and store, and can often be regularly replenished by the body. Several groups have supplemented the culture medium with synovial fluid in growing human, bovine, rabbit and dog chondrocytes, but mostly used only low levels of synovial fluid (below 20&#37;) 11-25. While chicken, horse and human chondrocytes have been cultured in the medium with higher percentage of synovial fluid, these culture systems were two-dimensional 26-28. Here we present our method of culturing human articular chondrocytes in a 3D system with a high percentage of synovial fluid (up to 100&#37;) over a period of 21 days. In doing so, we overcame a major hurdle presented by the high viscosity of the synovial fluid. This system provides the possibility of studying human chondrocytes in synovial fluid in a 3D setting, which can be further combined with two other important factors (oxygen tension and mechanical loading) 29,30 that constitute the natural environment for cartilage to mimic the natural milieu for cartilage growth. Furthermore, This system may also be used for assaying synovial fluid activity on chondrocytes and provide a platform for developing cartilage regeneration technologies and therapeutic options for arthritis.

A 3D system of culturing human articular chondrocytes in high levels of synovial fluid is described. Synovial fluid reflects the most natural microenvironment for articular cartilage, and can be easily obtained and stored. This system thus can be used for studying cartilage regeneration and for screening therapeutics for treating arthritis.

Cartilage destruction is a central pathological feature of osteoarthritis, a leading cause of disability in the US. Cartilage in the adult does not ...

Biology

A Friction Testing-Bioreactor Device for Study of Synovial Joint Biomechanics, Mechanobiology, and Physical Regulation

In primary osteoarthritis (OA), normal 'wear and tear' associated with aging inhibits the ability of cartilage to sustain its load-bearing and lubrication functions, fostering a deleterious physical environment. The frictional interactions of articular cartilage and synovium may influence joint homeostasis through tissue level wear and cellular mechanotransduction. To study these mechanical and mechanobiological processes, a device capable of replicating the motion of the joint is described. The friction testing device controls the delivery of reciprocal translating motion and normal load to two contacting biological counterfaces. This study adopts a synovium-on-cartilage configuration, and friction coefficient measurements are presented for tests performed in a phosphate-buffered saline (PBS) or synovial fluid (SF) bath. The testing was performed for a range of contact stresses, highlighting the lubricating properties of SF under high loads. This friction testing device can be used as a biomimetic bioreactor for studying the physical regulation of living joint tissues in response to applied physiologic loading associated with diarthrodial joint articulation.

The present protocol describes a friction testing device that applies simultaneous reciprocal sliding and normal load to two contacting biological counterfaces.

In primary osteoarthritis (OA), normal 'wear and tear' associated with aging inhibits the ability of cartilage to sustain its load-bearing and lubrication ...

Bioengineering

Creation of a Knee Joint-on-a-Chip for Modeling Joint Diseases and Testing Drugs

The high prevalence of debilitating joint diseases like osteoarthritis (OA) poses a high socioeconomic burden. Currently, the available drugs that target joint disorders are mostly palliative. The unmet need for effective disease-modifying OA drugs (DMOADs) has been primarily caused by the absence of appropriate models for studying the disease mechanisms and testing potential DMOADs. Herein, we describe the establishment of a miniature synovial joint-mimicking microphysiological system (miniJoint) comprising adipose, fibrous, and osteochondral tissue components derived from human mesenchymal stem cells (MSCs). To obtain the three-dimensional (3D) microtissues, MSCs were encapsulated in photocrosslinkable methacrylated gelatin before or following differentiation. The cell-laden tissue constructs were then integrated into a 3D-printed bioreactor, forming the miniJoint. Separate flows of osteogenic, fibrogenic, and adipogenic media were introduced to maintain the respective tissue phenotypes. A commonly shared stream was perfused through the cartilage, synovial, and adipose tissues to enable tissue crosstalk. This flow pattern allows the induction of perturbations in one or more of the tissue components for mechanistic studies. Furthermore, potential DMOADs can be tested via either &#34;systemic administration&#34; through all the medium streams or &#34;intraarticular administration&#34; by adding the drugs to only the shared &#34;synovial fluid&#34;-simulating flow. Thus, the miniJoint can serve as a versatile in vitro platform for efficiently studying disease mechanisms and testing drugs in personalized medicine.

We provide detailed methods for generating four types of tissues from human mesenchymal stem cells, which are used to recapitulate the cartilage, bone, fat pad, and synovium in the human knee joint. These four tissues are integrated into a customized bioreactor and connected through microfluidics, thus generating a knee joint-on-a-chip.

The high prevalence of debilitating joint diseases like osteoarthritis (OA) poses a high socioeconomic burden. Currently, the available drugs that target ...

Standardized Histomorphometric Evaluation of Osteoarthritis in a Surgical Mouse Model

One of the most prevalent joint disorders in the United States, osteoarthritis (OA) is characterized by progressive degeneration of articular cartilage, primarily in the hip and knee joints, which results in significant impacts on patient mobility and quality of life. To date, there are no existing curative therapies for OA able to slow down or inhibit cartilage degeneration. Presently, there is an extensive body of ongoing research to understand OA pathology and discover novel therapeutic approaches or agents that can efficiently slow down, stop, or even reverse OA. Thus, it is crucial to have a quantitative and reproducible approach to accurately evaluate OA-associated pathological changes in the joint cartilage, synovium, and subchondral bone. Currently, OA severity and progression are primarily assessed using the Osteoarthritis Research Society International (OARSI) or Mankin scoring systems. In spite of the importance of these scoring systems, they are semiquantitative and can be influenced by user subjectivity. More importantly, they fail to accurately evaluate subtle, yet important, changes in the cartilage during the early disease states or early treatment phases. The protocol we describe here uses a computerized and semiautomated histomorphometric software system to establish a standardized, rigorous, and reproducible quantitative methodology for the evaluation of joint changes in OA. This protocol presents a powerful addition to the existing systems and allows for more efficient detection of pathological changes in the joint.

The current protocol establishes a rigorous and reproducible method for quantification of morphological joint changes that accompany osteoarthritis. Application of this protocol can be valuable in monitoring disease progression and evaluating therapeutic interventions in osteoarthritis.

One of the most prevalent joint disorders in the United States, osteoarthritis (OA) is characterized by progressive degeneration of articular cartilage, ...

Flow Cytometry Analysis of Immune Cell Subsets within the Murine Spleen, Bone Marrow, Lymph Nodes and Synovial Tissue in an Osteoarthritis Model

Osteoarthritis (OA) is one of the most prevalent musculoskeletal diseases, affecting patients suffering from pain and physical limitations. Recent evidence indicates a potential inflammatory component of the disease, with both T-cells and monocytes/macrophages potentially associated with the pathogenesis of OA. Further studies postulated an important role for subsets of both inflammatory cell lineages, such as Th1, Th2, Th17, and T-regulatory lymphocytes, and M1, M2, and synovium-tissue-resident macrophages. However, the interaction between the local synovial and systemic inflammatory cellular response and the structural changes in the joint is unknown. To fully understand how T-cells and monocytes/macrophages contribute towards OA, it is important to be able to quantitively identify these cells and their subsets simultaneously in synovial tissue, secondary lymphatic organs and systemically (the spleen and bone marrow). Nowadays, the different inflammatory cell subsets can be identified by a combination of cell-surface markers making multi-color flow cytometry a powerful technique in investigating these cellular processes. In this protocol, we describe detailed steps regarding the harvest of synovial tissue and secondary lymphatic organs as well as generation of single cell suspensions. Furthermore, we present both an extracellular staining assay to identify monocytes/macrophages and their subsets as well as an extra- and intra-cellular staining assay to identify T-cells and their subsets within the murine spleen, bone marrow, lymph nodes and synovial tissue. Each step of this protocol was optimized and tested, resulting in a highly reproducible assay that can be utilized for other surgical and non-surgical OA mouse models.

Here, we describe a detailed and reproducible flow cytometry protocol to identify monocyte/macrophage and T-cell subsets using both extra- and intracellular staining assays within the murine spleen, bone marrow, lymph nodes and synovial tissue, utilizing an established surgical model of murine osteoarthritis.

Osteoarthritis (OA) is one of the most prevalent musculoskeletal diseases, affecting patients suffering from pain and physical limitations. Recent ...

Software-Assisted Quantitative Measurement of Osteoarthritic Subchondral Bone Thickness

Subchondral bone thickening and sclerosis are the major hallmarks of osteoarthritis (OA), both in animal models and in humans. Currently, the severity of the histologic subchondral bone thickening is mostly determined by visual estimation based semi-quantitative grading systems. This article presents a reproducible and easily executed protocol to quantitatively measure subchondral bone thickness in a mouse model of knee OA induced by destabilization of the medial meniscus (DMM). This protocol utilized ImageJ software to quantify subchondral bone thickness on histologic images after defining a region of interest in the medial femoral condyle and the medical tibial plateau where subchondral bone thickening usually occurs in DMM-induced knee OA. Histologic images from knee joints with a sham procedure were used as controls. Statistical analysis indicated that the newly developed quantitative subchondral bone measurement system was highly reproducible with low intra- and inter-observer variabilities. The results suggest that the new protocol is more sensitive to subtle or mild subchondral bone thickening than the widely used visual grading systems. This protocol is suitable for detecting both early and progressing osteoarthritic subchondral bone changes and for assessing in vivo efficacy of OA treatments in concert with OA cartilage grading.

This methodology article presents a software-assisted quantitative measurement protocol to quantify histologic subchondral bone thickness in murine osteoarthritic knee joints and normal knee joints as controls. This protocol is highly sensitive to subtle thickening and is suitable for detecting early osteoarthritic subchondral bone changes.

Subchondral bone thickening and sclerosis are the major hallmarks of osteoarthritis (OA), both in animal models and in humans. Currently, the severity of ...

Knee Arthrocentesis in Adults

Arthrocentesis of the knee is a procedure in which a needle is inserted into the knee joint, and synovial fluid is aspirated. An arthrocentesis can be diagnostic or therapeutic. Synovial fluid may be removed for testing to determine the nature of the knee effusion. If septic arthritis is suspected, urgent arthrocentesis before initiation of antibiotic treatment is indicated. Moreover, arthrocentesis can also aid in diagnosing crystal-induced arthritis such as gout or pseudogout, or non-inflammatory arthritis such as osteoarthritis. Identifying the cause of the knee effusion can guide treatment. Furthermore, removing fluid from a knee can reduce intraarticular pressure to decrease pain and improve range of motion. There is no absolute contraindication to performing this procedure, but in selecting the needle entry site, an area of skin that is infected should be avoided. Therefore, caution should be exercised when a patient presents with suspected cellulitis over the knee joint to avoid the potential risk of causing iatrogenic septic arthritis. A knee that has undergone arthroplasty should be assessed for arthrocentesis by an orthopedic surgeon. Arthrocentesis of the knee is typically performed with the patient supine. The site for needle insertion is marked, and then the skin is disinfected. After a local anesthetic is administered, a needle is inserted along the pathway that was anesthetized. Synovial fluid is aspirated, and then the needle is withdrawn. Pressure is applied until any bleeding stops. The synovial fluid can be analyzed for infection and inflammation but cannot directly confirm a diagnosis of internal derangement or autoimmune causes of arthritis. In addition to the history and physical examination, laboratory findings and imaging can clarify the etiology of a knee effusion.

Here the protocol describes arthrocentesis of the knee, a procedure in which a needle is inserted into the knee joint, and synovial fluid is aspirated. Synovial fluid may be removed for testing to determine the nature of the knee effusion. Arthrocentesis of the knee is typically performed with the patient supine.

Arthrocentesis of the knee is a procedure in which a needle is inserted into the knee joint, and synovial fluid is aspirated. An arthrocentesis can be ...

Destabilization of the Medial Meniscus and Cartilage Scratch Murine Model of Accelerated Osteoarthritis

Osteoarthritis is the most prevalent musculoskeletal disease in people over 45, leading to an increasing economic and societal cost. Animal models are used to mimic many aspects of the disease. The present protocol describes the destabilization and cartilage scratch model (DCS) of post-traumatic osteoarthritis. Based on the widely used destabilization of the medial meniscus (DMM) model, DCS introduces three scratches on the cartilage surface. The current article highlights the steps to destabilize the knee by transecting the medial meniscotibial ligament followed by three intentional superficial scratches on the articular cartilage. The possible analysis methods by dynamic weight-bearing, microcomputed tomography, and histology are also demonstrated. While the DCS model is not recommended for studies that focus on the effect of osteoarthritis on the cartilage, it enables the study of osteoarthritis development in a shorter time window, with special focus on (1) osteophyte formation, (2) osteoarthritic and injury pain, and (3) the effect of cartilage damage in the whole joint.

The present protocol describes the controlled microblade scratches on the surface of the articular cartilage after destabilizing the mouse knee by cutting the medial miniscotibial ligament. This animal model presents an accelerated form of osteoarthritis (OA) suitable for studying osteophyte formation, osteosclerosis, and early-stage pain.

Osteoarthritis is the most prevalent musculoskeletal disease in people over 45, leading to an increasing economic and societal cost. Animal models are ...

Ameliorating Osteoarthritis in Mice Using Silver Nanoparticles

Knee osteoarthritis (KOA) is one of the most commonly encountered degenerative diseases of the joints in people over 45 years of age. Currently, there are not any effective therapeutics for KOA,and the only end-point strategy is total knee arthroplasty (TKA); therefore, KOA is associated with economic burdens and societal costs. The immune inflammatory response is involved in the occurrence and development of KOA. We previously established a mouse model of KOA using type II collagen. Hyperplasia of the synovial tissue was present in the model, alongside a large number of infiltrated inflammatory cells. Silver nanoparticles have substantial anti-inflammatory effects and have been widely used in tumor therapy and surgical drug delivery. Therefore, we evaluated the therapeutic effects of silver nanoparticles in a collagenase II-induced KOA model. The experimental results showed that silver nanoparticles significantly reduced synovial hyperplasia and the infiltration of neutrophils in the synovial tissue. Hence, this work demonstrates the identification of a novel strategy for OA and provides a theoretical basis for preventing the progress of KOA.

Presented here is a protocol for using silver nanoparticles to effectively ameliorate the acute symptoms of type II collagenase-induced osteoarthritis mice, including synovial inflammation, synovial hyperplasia, vascular hyperplasia, etc.

Knee osteoarthritis (KOA) is one of the most commonly encountered degenerative diseases of the joints in people over 45 years of age. Currently, there are ...

Tissue Collection and RNA Extraction from the Human Osteoarthritic Knee Joint

Osteoarthritis (OA) is a chronic and degenerative joint disease most often affecting the knee. As there is currently no cure, total knee arthroplasty (TKA) is a common surgical intervention. Experiments using primary human OA tissues obtained from TKA provide the capability to investigate disease mechanisms ex vivo. While OA was previously thought to impact mainly the cartilage, it is now known to impact multiple tissues in the joint. This protocol describes patient selection, sample processing, tissue homogenization, RNA extraction, and quality control (based on RNA purity, integrity, and yield) from each of seven unique tissues to support disease mechanism investigation in the knee joint. With informed consent, samples were obtained from patients undergoing TKA for OA. Tissues were dissected, washed, and stored within 4 h of surgery by flash freezing for RNA or formalin fixation for histology. Collected tissues included articular cartilage, subchondral bone, meniscus, infrapatellar fat pad, anterior cruciate ligament, synovium, and vastus medialis oblique muscle. RNA extraction protocols were tested for each tissue type. The most significant modification involved the method of disintegration used for low-cell, high-matrix, hard tissues (considered as cartilage, bone, and meniscus) versus relatively high-cell, low-matrix, soft tissues (considered as fat pad, ligament, synovium, and muscle). It was found that pulverization was appropriate for hard tissues, and homogenization was appropriate for soft tissues. A proclivity for some subjects to yield higher RNA integrity number (RIN) values than other subjects consistently across multiple tissues was observed, suggesting that underlying factors such as disease severity may impact RNA quality. The ability to isolate high-quality RNA from primary human OA tissues provides a physiologically relevant model for sophisticated gene expression experiments, including sequencing, that can lead to clinical insights that are more readily translated to patients.

Primary tissues obtained from patients following total knee arthroplasty provide an experimental model for osteoarthritis research with maximal clinical translatability. This protocol describes how to identify, process, and isolate RNA from seven unique knee tissues to support mechanistic investigation in human osteoarthritis.

Osteoarthritis (OA) is a chronic and degenerative joint disease most often affecting the knee. As there is currently no cure, total knee arthroplasty ...

Synovial Fluid Analysis to Identify Osteoarthritis

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