Name: synovial fluid analysis to identify osteoarthritis
Uploaded: 2022-10-20T14:30:00
Description: Watch this Scientific Journal Video about Synovial Fluid Analysis to Identify Osteoarthritis at JoVE.com

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...

<ol>
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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...

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 can reveal the degree of inflammation and the presence of pathogenic crystals during the initial evaluation of patient with musculoskeletal symptoms and joint effusions. The main advantage of this technique is its simplicity and reliability. In a few steps, the clinician can obtain important diagnostic information on the patient's disease.The identification of specific crystals is vital for diagnosis and treatment. Urate crystals indicate gout, whereas calcium pyrophosphate and other calcium crystals indicate calcium-related arthropathies or a more severe form of osteoarthritis. To begin, take the synovial fluid sample collected during arthrocentesis of swollen joints of patients.Annotate the volume of the sample in milliliters and transfer it separately into a tube containing anticoagulant EDTA and another tube without any additive. In the suspected cases of infection, aseptically transfer a few milliliters of the synovial fluid sample into a culture bottle for microbiological testing. To examine the sample macroscopically, take a tube containing the sample and define the color of the sample, which can range from pale to dark yellow.Record the presence of blood, if any. Define the degree of clarity of the sample by observing it in the backlight and by determining the ease of reading a printed page through the tube containing the sample. Then assess the viscosity of the sample by dripping it from a disposable pipette.While the appearance of a long string of liquid indicates high viscosity, the formation of drops is indicative of low viscosity. Observe the sample through the tube with light in the background for the presence of any particulate materials including tissue fragments or fibrin. To determine the total white blood cell count, draw 0.5 microliters of the sample from the tube containing EDTAA using a Malassez-Potain pipette.Use the same pipette to draw 9.5 microliters of 0.1%methylene blue solution. Mix the sample and methylene blue solution in the pipette by gentle inversion. Discard the first few drops before pouring the mixture into the counting chamber of a hemocytometer.Now count the cells in two consecutive squares out of nine and multiply the obtained number by 100. Express the total white blood cell count as the number of leukocytes per cubic millimeter. Use the synovial fluid sample from the tube without any additive to determine the differential white blood cell count of the synovial fluid samples.To determine the differential cell count using supravital staining, take a ready-to-use slide pre-coated with crystal violet and methylene blue and place one small drop of sample in the center of the slide. Next, cover the sample drop with a coverslip and place a drop of microscope immersion oil on the coverslip. To use traditional staining for estimating the differential cell count, begin by putting a small drop of the sample at the end of a clean slide.Now use a second slide or a coverslip to spread the drop along the slide with a forward movement. Let the smear dry in the air. Examine the slide containing the air dried smear under 1, 000X oil immersion objective.Estimate the differential cell count by counting 100 consecutive cells while distinguishing the polymorphonuclear cells, the monocytes, and the lymphocytes from one another. Clean a glass slide with optical or soft paper imbibed with ethanol. Apply a small drop of the sample not mixed with any additive on the glass slide and cover it with a coverslip before placing the slide under the microscope.Focus the slide under the microscope using a low power magnification objective and carefully examine it under a bright field with a 40X objective. Look inside and outside the cells to identify the crystals according to their size and shape, which may be rhomboidal, needle shaped, or rod shaped. Then insert the polarizing filter between the light source and the specimen.To create a dark background, rotate the polarizer until its optical axis is perpendicular to the analyzer. 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. Observe the color revealed by the crystal.For Alizarin Red staining, mix a drop of the synovial fluid sample with an equal volume of previously prepared Alizarin Red solution on a clean slide. Cover the slide with a coverslip and examine at 400X magnification under a microscope to identify the crystals. The macroscopic characteristics of synovial fluid samples indicated the degree of inflammation.A sample with pale yellow color and more clarity is generally associated with non-inflammatory conditions such as osteoarthritis. On the other hand, a sample having dark yellow color and a turbid appearance indicates the presence of cells, debris, hyaluronic acid, and fibrin fragments which are associated with inflammatory effusions. The leukocytes observed in synovial fluid samples from osteoarthritis patients were mostly monocytes and other mononuclear cells like synoviocytes.Calcium pyrophosphate crystals were detected in synovial fluid samples indicating relatively higher levels of inflammatory cytokines. Moreover, a positive Alizarin Red staining revealed the presence of basic calcium phosphate crystals in the samples. It is very important to carefully examine the slide under ordinary light when searching for crystals.Remember that pyrophosphate crystals are often weakly birefringent and can be missed under polarized light. The presence of calcium crystals in synovial fluid and their association with more severe form of disease have led researchers to explore new mechanisms in the pathogenesis and the progression of osteoarthritis.

Research

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Medicine

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