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.
