Synovial fluid analysis

INTRODUCTION — Synovial fluid analysis is helpful for determining the underlying cause of arthritis, particularly for septic or crystal-induced arthritis. The white cell count, differential count, cultures, Gram stain, and crystal search using polarized light microscopy are the most useful studies. An overview of synovial fluid analysis in the native joint is discussed here. Septic arthritis, prosthetic joint infections, gonococcal arthritis, and fungal and mycobacterial arthritis are discussed in detail separately. (See "Septic arthritis in adults" and "Prosthetic joint infection: Epidemiology, microbiology, clinical manifestations, and diagnosis" and "Prosthetic joint infection: Treatment" and "Disseminated gonococcal infection" and "Bone and joint tuberculosis".)

INDICATIONS — Arthrocentesis with synovial fluid analysis should be attempted in all patients who have a joint effusion or signs suggestive of inflammation within the joint, without a known cause. The most important reason to perform a synovial fluid analysis may be to evaluate for septic arthritis in a patient presenting with an acutely swollen joint that is painful and warm even in patients with a known arthritic disease. (See "Septic arthritis in adults".)

Synovial fluid analysis is also particularly important for definitively diagnosing gout or pseudogout. In combination with a negative Gram stain and bacterial cultures, a diagnosis of gout or pseudogout may be established by confirming the presence of crystals of monosodium urate (MSU) or calcium pyrophosphate dihydrate (CPPD) in a patient presenting with unexplained inflammatory arthritis. (See "Clinical manifestations and diagnosis of gout" and "Clinical manifestations and diagnosis of calcium pyrophosphate crystal deposition (CPPD) disease".)

Synovial fluid analysis is also helpful in narrowing the differential diagnosis by distinguishing between categories of joint effusions (ie, inflammatory versus noninflammatory) (table 1), and to diagnose hemarthrosis (algorithm 1). (See 'Categories of joint effusions' below.)

CATEGORIES OF JOINT EFFUSIONS — Results of synovial fluid analysis can be used to categorize the fluid as noninflammatory, inflammatory, septic, or hemorrhagic based upon the clinical and laboratory analysis (table 2).

●Noninflammatory – Synovial fluid with a nucleated (white) cell count of approximately <2000 white blood cells (WBCs)/mm³ is noninflammatory. Examples of conditions associated with noninflammatory synovial fluid include osteoarthritis, avascular necrosis, or a meniscal tear.

●Inflammatory – Synovial fluid with a nucleated cell count ≥2000 WBCs/mm³ is inflammatory in the absence of blood, either as a "bloody tap" or hemarthrosis. A wide range of conditions are associated with inflammatory synovial fluid such as septic arthritis, crystal-induced arthritis (eg, gout, pseudogout), or spondyloarthritis. The range of nucleated (white) cell counts that can be observed with each of these conditions overlap considerably [1].

●Septic – Septic effusions may be due to bacteria, mycobacteria, or fungus. Septic effusions have an inflammatory white nucleated cell count, with ≥2000 WBCs/mm³. There is no specific cutoff for septic arthritis, but the likelihood of septic arthritis increases with the observed synovial fluid white cell count.

●Hemorrhagic – Large numbers of red blood cells (RBCs) give synovial fluid the appearance of being hemorrhagic. They may be due to hemophilia, anticoagulation or other hemorrhagic diathesis, scurvy, trauma (with or without fracture), neuropathic arthropathy, and tumor (including pigmented villonodular synovitis, synovioma, hemangioma, and other benign or malignant neoplasms). (See "Overview of hemarthrosis".)

Accidental trauma to a capillary or venule may occur during the performance of a joint aspiration, resulting in an elevated RBC count in the fluid. When traumatic joint aspirations occur, the release of blood into synovial fluid may raise the WBC count (1 WBC for every 500 to 1000 RBCs). Some clinicians adjust the synovial fluid WBC count to account for the presence of RBCs that have entered the sample by the trauma of the aspiration [2].

Note that overtly bloody fluid (ie, hemarthrosis) may have cell counts similar to that of the peripheral blood, with a RBC:WBC ratio of approximately 500 to 1000:1.

Characteristic synovial fluid abnormalities result from specific pathologic mechanisms which underlie the accumulation of joint fluid. As an example, when the synovial membrane becomes inflamed, there is an influx of WBCs into the synovial tissue; the synovial membrane subsequently becomes leaky, thereby resulting in the exudation of plasma proteins into the synovial fluid.

The differential diagnosis of each of these specific categories is broad and not necessarily exclusive.

SPECIMEN COLLECTION AND HANDLING — Aspiration is performed under aseptic conditions with the specimen quickly transferred to sterile tubes and plated as soon as possible. A purple-top (EDTA) or green-top (heparin) tube is used by the laboratory for cell count and crystal analysis, and a sterile tube should be used for culture. If there is sufficient fluid set aside for Gram stain and routine culture, joint fluids should have an aliquot injected into an aerobic blood culture bottle for improved culture yield [3]. When anaerobic bacteria are suspected, anaerobic blood culture bottles should be used. (See 'Bacterial culture' below.)

Techniques for aspirating selected joints in adults and children are described separately. (See "Joint aspiration or injection in adults: Technique and indications" and "Joint aspiration or injection in children: Indications, technique, and complications".)

Care must be taken not to contaminate the synovial fluid sample with injectable corticosteroid solutions, since steroid crystals may make the search for other crystals and the assessment of the Gram stain difficult.

Inflammatory joint fluids often contain increased amounts of fibrinogen and fibrin and may clot during transport to the laboratory or while awaiting testing. Obtaining an accurate count of nucleated cells in synovial fluid is facilitated by placing at least 1 mL of fluid in an anticoagulant-containing tube, either EDTA-containing or heparinized [4]. If there is too little fluid to allow its transfer into a tube containing an anticoagulant, expedited transport of a drop of fresh synovial fluid on a slide with a cover slip to, and handling by, the laboratory may improve the likelihood of a technically satisfactory cell count. Examination of 10 high-power fields and the finding that there are zero to two nucleated cells per field is correlated with nucleated cell counts of <1300/mm³ [5]. Cell count and crystal search should be performed promptly as disintegration of cells and dissolution of crystals occurs over time [6-8]. The cell count declines within a matter of hours [6]. A couple of studies suggest that monosodium urate (MSU) crystals and calcium pyrophosphate (CPP) crystals remain detectable up to three days or more after the arthrocentesis; however, the quantity of crystals decline over time with CPP crystals deteriorating faster than MSU crystals [7,8]. Artifactual crystals can also form over time, confusing the results. (See 'Nucleated (white) cell count and differential' below and 'Crystal analysis' below.)

Use of talc-free gloves is recommended when preparing synovial fluid for a crystal search, as contamination of the slide with birefringent talc particles may make the microscopic examination for pathogenetically important crystals more difficult [9].

Microscopic examination — The microscopic examination of synovial fluid may be performed on as little as one drop of uncentrifuged fluid (wet mount); however, examination of the sediment of a centrifuged specimen may improve the sensitivity of the microscopic examination for crystals [10]. The sediment is also more adherent to glass microscope slides than unspun synovial fluid and may provide a better sample for preparation of the Gram stain [11,12].

The Gram stain is typically performed by laboratory personnel and is discussed further below (see 'Gram stain' below). The crystal analysis can be performed at the point of care if the clinician's office is equipped with a polarized light microscope with a red compensator. (See 'Crystal analysis' below.)

ROUTINE COMPONENTS OF SYNOVIAL FLUID ANALYSIS — Routine synovial fluid analysis should include gross inspection of the synovial fluid to evaluate clarity, color, and viscosity as well as microscopic assessment for Gram stain, cell count, and crystal detection (algorithm 1).

Gross appearance — The volume, clarity, color, and viscosity of joint fluid should be noted. Normal joints contain a small volume of synovial fluid that is highly viscous, clear, and essentially acellular.

●Clarity – Increased opacity of the fluid is usually due to abnormally large numbers of nucleated or red blood cells (RBCs). However, translucent or even opaque fluid may be the result of acellular material. Examples include lipids in fat necrosis, chyle droplets, or innumerable monosodium urate (MSU) crystals aspirated from gouty tophi.

●Color – Colorless, clear fluid is normal, while increasing amounts of plasma and nucleated cells contribute to the yellow or yellow-green appearance of inflammatory or septic fluids. Bright red, rusty, or chocolate-brown fluids are indicative of fresh or old blood.

●Viscosity – As joint fluid is expelled from the syringe and allowed to drop into a suitable receptacle, normal fluid will produce a long string-like extension as it falls. Release of proteolytic enzymes into inflamed synovial fluid typically causes a decrease in viscosity. However, frankly purulent (septic) effusions may also be viscous.

Crystal analysis — Synovial fluid should be examined for MSU crystals and calcium pyrophosphate dihydrate (CPPD) crystals to evaluate for gout or pseudogout, respectively. Examination of synovial fluid for crystals is facilitated by using a microscope with polarizing filters and a quarter-wave plate (also known as a "red compensator"). (See "Clinical manifestations and diagnosis of gout" and "Clinical manifestations and diagnosis of calcium pyrophosphate crystal deposition (CPPD) disease".)

Birefringence is a term used to describe the optical property associated with certain transparent crystals in which the speed of propagation of light along the major and minor axes of the crystal differs, causing the plane of polarized light to be rotated. Detection of birefringent crystals is facilitated by use of two plane-polarizing filters, one between the light source and the sample and the other between the sample and the observer's eye. When the polarized filters are crossed, the background appears dark and birefringent material, including MSU and CPPD crystals, appear brighter than the background.

The red compensator is used to determine the direction of birefringence. Microscopes equipped with a red compensator will have the axis used for crystal analysis marked with an "S," "Z," or similar symbol indicating the "slow" axis. Birefringent crystals appear blue or yellow depending upon their "sign" of birefringence (negative or positive) and upon their orientation with respect to the axis of the analyzer.

Crystals of MSU and CPPD may be present in the same synovial fluid specimen [13]. They have also been noted within the same cell [10]. Synovial fluid should be examined within 24 to 48 hours as crystals (especially CPPD) will deteriorate over time, although refrigeration may slow this process [14].

An initial survey using a low-power objective lens can give a rapid assessment of whether or not crystals are present and may identify areas of the specimen for closer inspection with high-power magnification (eg, 400-fold). This technique is more effective for bright MSU crystals compared with CPPD crystals.

Monosodium urate crystals — MSU crystals are brightly birefringent when viewed with crossed polarized filters and are needle-shaped. When the red compensator is introduced into the light path and is adjusted so that the background appears red, MSU crystals appear yellow when aligned parallel to the "S" or "Z" axis indicated on the analyzer and blue when perpendicular (picture 1). MSU crystals are referred to as being "negatively birefringent."

Well-trained observers are able to accurately identify crystals if any are present [15]. However, substantial variability has been noted among hospital laboratories in the ability to properly identify the presence or absence of MSU and CPPD crystals in synovial fluids [16]. Studies of the performance of different hospital laboratories on the same synovial fluids suggest that MSU crystals are more easily detected than CPPD crystals [16].

The reported sensitivity of routine laboratory crystal analysis for MSU crystals ranges from 63 to 78 percent, specificity ranges from 93 to 100 percent, and positive likelihood ratio (+LR) is 14 for a diagnosis of gout [16].

Calcium pyrophosphate dihydrate crystals — CPPD crystals have a rhomboidal or rectangular shape and positive birefringence. However, compared with MSU crystals, the birefringence is weaker, and some CPPD crystals may not appear birefringent [17]. A birefringent CPPD crystal appears blue when its long axis is parallel to the "S" or "Z" axis indicated on the analyzer (picture 2).

Routine laboratory sensitivity for CPPD crystals ranges from 12 to 83 percent, specificity ranges from 78 to 96 percent, and positive LR is 2.9 for a diagnosis of CPPD-associated arthritis [16]. Many CPPD crystals are not birefringent, making their detection more difficult.

Other crystals — Crystals other than MSU and CPPD can also be found sometimes in synovial fluid. Examples of other crystals include crystals of cholesterol and basic calcium phosphate, including hydroxyapatite. Calcium-containing crystals other than CPPD are generally too small to detect with optical microscopy; however, chunks of massive basic calcium phosphate as non-birefringent shiny aggregates can be seen. Rarely, other crystals (ie, monoclonal proteins) may be seen. Their analysis may be confirmed by x-ray crystallography and electron microscopy.

●Lipids – Cholesterol crystals are sometimes noted in chronic inflammatory synovial fluids. Cholesterol crystals often appear as notched polygonal plates (picture 3). In contrast to cholesterol, some neutral fat (lipid) droplets have a "Maltese cross" appearance when viewed with polarizing microscopy that is identical to that of lipid droplets seen in the urine sediment of patients with nephrotic syndrome (picture 4). When lipid droplets are present in synovial fluid, articular fracture should be suspected [18].

●Apatite (hydroxyapatite) crystals – Among the many techniques that have been used to detect apatite crystals [19], only light microscopy with alizarin red or von Kossa stains, which have an affinity for calcium-containing crystals, are considered to be practical for clinical use [10]. However, the sensitivity and specificity of microscopy using one or the other of these calcium stains is uncertain.

Nucleated (white) cell count and differential — The nucleated (white) cell count allows effusions to be classified as either noninflammatory or inflammatory (table 1). Substantial inter-laboratory variability has been noted in reported cell counts on aliquots of the same specimen [16]. In the case of accidental trauma to a capillary or venule during joint aspiration resulting in an elevated RBC count in the fluid, a calculated adjustment to the white cell count may be appropriate and is discussed further above. (See 'Categories of joint effusions' above.)

●Nucleated cell count – Normal synovial fluid is nearly acellular. Inflammatory and septic synovial fluids are characterized by increasing numbers of leukocytes. Bacterial joint infections typically are purulent with leukocyte counts (most of which are neutrophils) of 50,000 to 150,000 cells/mm³. However, using 50,000 white cells/mm³ as a cutoff for septic arthritis is not accurate, as was shown by a retrospective analysis reporting a sensitivity of 61 percent among patients with septic arthritis confirmed by synovial fluid culture growth of a pathogenic organism [20].

A 2007 systematic review of the predictive ability of diagnostic tests for septic arthritis in adults presenting with acute mono- or oligoarthritis concluded that the likelihood of septic arthritis increases with the observed synovial fluid white cell count [21]. In adults, as the synovial leukocyte count increased from 25,000/mm³ to over 50,000/mm³ or to over 100,000/mm³, the positive likelihood ratio (+LR) increased from 2.9 to 7.7 and to 28, respectively. Conversely, the likelihood of septic arthritis is modestly reduced in the setting of a synovial leukocyte count of less than 25,000/mm³ (estimated +LR 0.32) [21]. However, lower cell counts may be observed among immunocompromised patients with septic arthritis and in infections due to mycobacterial, some Neisseria, and several Gram-positive organisms.

●Nucleated cell differential – In normal synovial fluid or in noninflammatory joint effusions, polymorphonuclear (PMN) leukocytes represent a small proportion of the nucleated cells present. Inflammatory and septic fluids have increasing proportions of PMNs present. Bacterial joint infections often have ≥75 percent PMNs [22]. (See "Septic arthritis in adults".)

Eosinophilia in the synovial fluid suggests parasitic infection, allergy, neoplasm, or Lyme disease [23,24]. Other unusual features may also be apparent on the smear used for the differential cell count, including phagocytosis of nuclear material and the lupus erythematosus-cell phenomenon.

Malignant appearing cells may be noted in synovial fluid [18]. However, if there is a suspicion of joint involvement by a neoplasm or hematologic malignancy, formal cytologic examination is appropriate.

Gram stain — The synovial fluid Gram stain is an easily performed test that can provide immediate, useful information concerning the diagnosis and therapy (Gram-positive versus Gram-negative coverage) of septic arthritis. In addition to identifying common organisms, the Gram stain may be the only evidence of infection with fastidious organisms that are not able to grow in culture.

The sensitivity of the Gram stain varies from 29 to 50 percent; thus, a negative Gram stain does not rule out an infection, particularly if early or in infections due to certain microorganisms such as mycoplasma, mycobacteria, and fungi [21]. Gram stains of swabs obtained at the time of joint exploration (as is often the case in septic arthritis associated with prosthetic joints) are inferior to Gram stains of aspirated or collected joint fluid.

In some cases, the Gram stain results are classic and easy to identify, as with multiple Gram-positive cocci aspirated from an acutely swollen knee. However, common problems include:

●Clumps of stain or cellular material and other debris simulating bacteria.

●Incorrect blanching so that Gram-positive organisms appear Gram-negative or, rarely, vice versa.

●The presence of crystals in the synovial fluid. Although this finding is often indicative of crystal-induced arthritis (as in gout), some patients have, as noted above, coexisting crystal and septic arthritis [25].

It is, therefore, important that the clinician be familiar with the Gram stain technique and interpretation or at least should have access to laboratory facilities and personnel proficient in this task.

Bacterial culture — Synovial fluid should be sent for bacterial culture in a sterile tube; antibiotics should be withheld prior to joint aspiration. Additional testing for other infectious causes of arthritis are discussed separately. (See "Septic arthritis in adults", section on 'Differential diagnosis'.)

Common nongonococcal causes of bacterial arthritis are easily grown on routine culture media. As described above, the diagnostic yield is improved by inoculation of aerobic blood culture bottles [26,27] (see 'Specimen collection and handling' above and "Microbiology specimen collection and transport"). The microbiology of septic arthritis in children and adults are discussed in detail separately. (See "Septic arthritis in adults", section on 'Microbiology' and "Bacterial arthritis: Epidemiology, pathogenesis, and microbiology in infants and children", section on 'Microbiology'.)

SUMMARY AND RECOMMENDATIONS

●Indications – Synovial fluid analysis may be diagnostic in patients with bacterial joint infection and crystal-induced arthritis. This analysis is indicated in febrile patients with an acute flare of already established arthritis and in other situations in which the cause of a joint effusion is uncertain or septic arthritis is suspected. (See 'Indications' above.)

●Aspiration and injection techniques – Technical aspects of synovial fluid aspiration in adults and children are presented separately. (See "Joint aspiration or injection in adults: Technique and indications" and "Joint aspiration or injection in children: Indications, technique, and complications".)

●Categories of joint effusion – We suggest categorizing abnormal synovial fluids into those that are noninflammatory, inflammatory, septic, and hemorrhagic as a means to reduce the number of possible causes of effusions to consider in the differential diagnosis (table 2). However, for each category, there is significant diagnostic overlap. (See 'Categories of joint effusions' above.)

●Routine components of synovial fluid analysis – The most valuable components of laboratory analysis of synovial fluid are the nucleated (white) cell count, differential count, cultures, Gram stain, and crystal search using polarized light microscopy (algorithm 1). (See 'Routine components of synovial fluid analysis' above.)

•Gross appearance – The volume, clarity, color, and viscosity of joint fluid should be noted. Normal joints contain a small volume of synovial fluid that is highly viscous, clear, and essentially acellular. (See 'Gross appearance' above.)

•Crystal analysis – Synovial fluid should be examined for monosodium urate (MSU) crystals and calcium pyrophosphate dihydrate (CPPD) crystals to evaluate for gout or pseudogout, respectively. (See 'Crystal analysis' above.)

•Nucleated (white) cell count and differential – The nucleated (white) cell count allows effusions to be classified as either noninflammatory or inflammatory (table 1). (See 'Nucleated (white) cell count and differential' above.)

•Gram stain – The synovial fluid Gram stain is an easily performed test that can provide immediate, useful information concerning the diagnosis and therapy (Gram-positive versus Gram-negative coverage) of septic arthritis. In addition to identifying common organisms, the Gram stain may be the only evidence of infection with fastidious organisms that are not able to grow in culture. (See 'Gram stain' above.)

•Bacterial culture – Synovial fluid should be sent for bacterial culture; antibiotics should be withheld prior to joint aspiration. Additional testing for other infectious causes of arthritis are discussed separately. (See 'Bacterial culture' above and "Septic arthritis in adults", section on 'Differential diagnosis'.)