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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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&#160;</td>
		</tr>
		<tr>
			<td>EDTA tubes</td>
			<td>BD</td>
			<td>368861</td>
			<td>For SF collection&#160;</td>
		</tr>
		<tr>
			<td>Glass slides&#160;</td>
			<td>Merck</td>
			<td>S8902</td>
			<td>For crystal search</td>
		</tr>
		<tr>
			<td>Lambda filter (compensator)</td>
			<td>Any&#160;</td>
			<td>Refer to microscope company</td>
			<td>For crystal identification&#160;</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&#160;</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&#160;</td>
			<td>Refer to microscope company</td>
			<td>For crystal identification&#160;</td>
		</tr>
		<tr>
			<td>Testsimplet&#160;</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...

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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 ...

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4.0K Views.  University of Padova. 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.

Video: Synovial Fluid Analysis to Identify Osteoarthritis

The Monoiodoacetate Model of Osteoarthritis Pain in the Mouse

A major symptom of patients with osteoarthritis (OA) is pain that&#160;is triggered by peripheral as well as central changes within the pain pathways. The current treatments for OA pain such as NSAIDS or opiates are neither sufficiently effective nor devoid of detrimental side effects. Animal models of OA are being developed to improve our understanding of OA-related pain mechanisms and define novel pharmacological targets for therapy. Currently available models of OA in rodents include surgical and chemical interventions into one knee joint. The monoiodoacetate (MIA) model has become a standard for modelling joint disruption in OA in both rats and mice. The model, which is easier to perform in the rat, involves injection of MIA into a knee joint that induces rapid pain-like responses in the ipsilateral limb, the level of which can be controlled by injection of different doses. Intra-articular injection of MIA disrupts chondrocyte glycolysis by inhibiting glyceraldehyde-3-phosphatase dehydrogenase and results in chondrocyte death, neovascularization, subchondral bone necrosis and collapse, as well as inflammation. The morphological changes of the articular cartilage and bone disruption are reflective of some aspects of patient pathology. Along with joint damage, MIA injection induces referred mechanical sensitivity in the ipsilateral hind paw and weight bearing deficits that are measurable and quantifiable. These behavioral changes resemble some of the symptoms reported by the patient population, thereby validating the MIA injection in the knee as a useful and relevant pre-clinical model of OA pain.
The aim of this article is to describe the methodology of intra-articular injections of MIA and the behavioral recordings of the associated development of hypersensitivity with a mind to highlight the necessary steps to give consistent and reliable recordings.

Osteoarthritis (OA), or degenerative joint disease, is a debilitating condition associated with pain that remains only partially controlled by available analgesics. Animal models are being developed to improve our understanding of OA-related pain mechanisms. Here we describe the methodology for the monoiodoacetate model of OA pain in the mouse.

A major symptom of patients with osteoarthritis (OA) is pain that&#160;is triggered by peripheral as well as central changes within the pain pathways. The ...

Isolation and Cellular Phenotyping of Mesenchymal Stem Cells Derived from Synovial Fluid and Bone Marrow of Minipigs

Mesenchymal stem cells (MSCs) have been established after isolation from various tissue sources, including bone marrow and synovial fluid. Recently, synovial-fluid-derived MSCs were reported to have multi-lineage differentiation potential and immunomodulatory features, which indicates that these cells can be used for tissue engineering and systemic treatments. This study presents a protocol for simple and non-invasive isolation of MSCs derived from the bone marrow and synovial fluid of minipigs to analyze cell surface markers for cell phenotyping and in vitro culturing. Using sexually mature six-month-old minipigs, bone marrow was extracted from the iliac crest bone using a bone marrow extractor, and the synovial fluid was aspirated from the femorotibial joint. Procedures for the collection of samples from both sources were non-invasive. The protocols for effective isolation of MSCs from harvested cell sources and for creating in vitro culture conditions to expand stable MSCs from minipigs and the application of systemic autologous treatments are provided. For cell phenotyping, the cell surface markers of both cells were analyzed using flow cytometry. In the results, the MSCs were isolated from the synovial fluid of the minipigs and showed that synovial-fluid-derived MSCs have a similar morphology and cell phenotype to bone-marrow-derived MSCs. Therefore, non-invasively obtained synovial fluid is a valuable source of MSCs.

A protocol establishing mesenchymal stem cells (MSCs) isolated from the synovial fluid and bone marrow of minipigs and non-invasively collected using syringe aspiration is presented. Cellular phenotyping was performed using flow cytometry after cell isolation and in vitro cultivation of bone marrow and synovial fluids derived from MSCs.

Mesenchymal stem cells (MSCs) have been established after isolation from various tissue sources, including bone marrow and synovial fluid. Recently, ...

The Lower Body Positive Pressure Treadmill for Knee Osteoarthritis Rehabilitation

Here, based on a clinician&rsquo;s point-of-view, we propose a two-model lower body positive pressure (LBPP) protocol (walking and squatting models) in addition to a clinical, functional assessment methodology, including details for further encouragement of the development of non-drug surgical intervention strategies in knee osteoarthritis patients. However, we only present the effect of LBPP training in improvement of pain and knee function in one patient through three-dimensional gait analysis. The exact, long-term effects of this approach should be explored in future studies.

Here, based on a clinician&#8217;s point-of-view, we propose a two-model lower body positive pressure (LBPP) protocol (walking and squatting models) in addition to a clinical, functional assessment methodology, including details for further encouragement of the development of non-drug surgical intervention strategies in knee osteoarthritis patients. However, we only present the effect of LBPP training in improvement of pain and knee function in one patient through three-dimensional gait analysis. The exact, long-term effects of this approach should be explored in future studies.

Here, based on a clinician&rsquo;s point-of-view, we propose a two-model lower body positive pressure (LBPP) protocol (walking and squatting models) in ...

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 ...

Evaluation of Fluid Overload by Bioelectrical Impedance Vectorial Analysis

Early detection and management of fluid overload are critically important in acute illness, as the impact of therapeutic intervention can result in decreased or increased mortality rates. Accurate fluid status assessment entails appropriate therapy. Unfortunately, as the gold standard method of radioisotopic fluid measurement is costly, time-consuming, and lacks sensitivity in the acute care clinical setting, other less-accurate methods are typically used, such as clinical examination or 24 h output. Bioelectrical impedance vectorial analysis (BIVA) is an alternative impedance-based approach, where the raw parameter resistance and reactance of a subject are plotted to produce a vector, the position of which can be evaluated relative to tolerance intervals in an R-Xc graph. The fluid status is then interpreted as normal or abnormal, based on the distance from the mean vector derived from a healthy reference population. The objective of the present study is to demonstrate how to evaluate the presence of fluid overload through bioelectrical impedance vectorial analysis and the impedance ratio measured with tetrapolar multi-frequency equipment in patients admitted to the emergency department.

In this study, we demonstrate how to evaluate the presence of fluid overload through bioelectrical impedance vectorial analysis (BIVA) and the impedance ratio measured using tetrapolar multi-frequency equipment in patients admitted to the emergency department. BIVA and impedance ratio are reliable and useful tools to predict poor outcomes.

Early detection and management of fluid overload are critically important in acute illness, as the impact of therapeutic intervention can result in ...

FSN Therapy for Knee Osteoarthritis Pain Management

Fu's Subcutaneous Needling for Knee Osteoarthritis Pain

Fu's subcutaneous needling (FSN) is a new acupuncture and dry needling technique based on traditional Chinese medicine. It rapidly produces long-lasting effects in soft tissue injuries, particularly in painful musculoskeletal conditions, by providing stimulation primarily in the subcutaneous area. Osteoarthritis (OA) is the most common joint disease in adults worldwide and is often accompanied by a painful syndrome of structural changes in the peripheral joints of the knee. However, the etiology of OA pain is not fully understood, though myofascial trigger points (MTrPs) are commonly found in the lower limb muscles (so-called "tightened muscles") of patients with knee OA. 
FSN has been used in many fields for the treatment of acute pain problems and can relieve muscle contraction from MTrPs, thereby improving the local circulation. This study recruited patients with pain from knee OA into an FSN group or a transcutaneous electrical nerve stimulation (TENS) group with three treatment sessions and a follow-up over the course of 2 weeks. The results showed that FSN was effective in treating soft tissue pain around the knee with OA. This study aimed to establish and visualize three key technical indicators during FSN therapy, including the FSN needle insertion point and layer; the frequency and duration of the swaying movement; and the manipulation of the reperfusion approach. These findings have great potential for future applications in myofascial pain treatment, especially for pain management. Following this protocol could enhance FSN skills.

Author Spotlight: Fu's Subcutaneous Needling for Knee Osteoarthritis Pain  

We present a protocol for using Fu's subcutaneous needling for knee osteoarthritis pain, which combines swaying movement and reperfusion approach techniques. This protocol has great potential for future applications in myofascial pain treatment and could enhance Fu's subcutaneous needling (FSN) manipulation skills.

Fu's subcutaneous needling (FSN) is a new acupuncture and dry needling technique based on traditional Chinese medicine. It rapidly produces long-lasting ...

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 ...

Standardized Tuina Manipulation for Knee Osteoarthritis in Rats

Tuina Manipulation to Reduce Inflammation and Cartilage Loss in Knee Osteoarthritis Rats

Clinical trials suggest that Tuina manipulation is effective in treating knee osteoarthritis (KOA), while further studies are required to discover its mechanism. Therefore, the manipulation of animal models of knee osteoarthritis is critical. This protocol provides a standard process for Tuina manipulation on KOA rats and a preliminary exploration of the mechanism of Tuina for KOA. The press and kneading manipulation method (a kind of Tuina manipulation that refers to pressing and kneading the specific area of the body surface) is applied on 5 acupoints around the knee joint of rats. The force and frequency of the manipulation were standardized by finger pressure recordings, and the position of the rat during manipulation is described in detail in the protocol. The effect of manipulation can be measured by pain behavior tests and microscopic findings in synovial and cartilage. KOA rats showed significant improvement in pain behavior. The synovial tissue inflammatory infiltration was reduced in the Tuina group, and the expression of tumor necrosis factor (TNF)-&#945; was significantly lower. Compared to the control group, chondrocyte apoptosis was less in the Tuina group. This study provides a standardized protocol for Tuina manipulation on KOA rats and preliminary proof that the therapeutic effects of Tuina may be related to reducing synovial inflammation and delayed chondrocyte apoptosis.

Author Spotlight: Understanding the Mechanism of Tuina Manipulation in Rats 

We present a protocol for Tuina manipulation performed on plaster-immobilized-induced knee osteoarthritis rats. Based on the preliminary results, it suggested that the efficacy of the method relied on the reduction of inflammation and cartilage loss.

Clinical trials suggest that Tuina manipulation is effective in treating knee osteoarthritis (KOA), while further studies are required to discover its ...

Treating Knee Osteoarthritis with Tuina in a Rabbit Model

Tuina Intervention in Rabbit Model of Knee Osteoarthritis

Knee osteoarthritis (KOA) is mainly characterized by degenerative changes in the knee joint's cartilage and surrounding soft tissues. The efficacy of Tuina in treating KOA has been confirmed, but the underlying mechanism needs to be investigated. This study aims to establish a scientifically feasible KOA rabbit model treated with Tuina to reveal the underlying mechanisms. For this, 18, 6-month-old normal-grade male New Zealand rabbits were randomly divided into sham, model, and Tuina groups, with 6 rabbits in each group. The KOA model was established by injecting 4% papain solution into the knee joint cavity. The Tuina group was intervened with Tuina combined with the knee joint rotary correction method for 4 weeks. Only the standard grasping and fixation were performed in sham and model groups. At the end of the 1-week intervention, the knee joint range of motion (ROM) was observed, and cartilage hematoxylin-eosin (HE) staining was done. The study shows that Tuina could inhibit chondrocyte apoptosis, repair cartilage tissue, and restore knee joint ROM. In conclusion, this study demonstrates the scientific feasibility of Tuina treatment for KOA model rabbits, highlighting its potential application in the study of KOA and similar knee joint-related conditions.

Author Spotlight: Using a Rabbit Model to Explore the Efficacy of Tuina in Treating Knee Osteoarthritis 

The protocol describes a method for Tuina intervention in a rabbit model of knee osteoarthritis.

Knee osteoarthritis (KOA) is mainly characterized by degenerative changes in the knee joint's cartilage and surrounding soft tissues. The efficacy of ...