On Synovial Fluid Cristallography Training for Arthrocentesis Performing Physicians

Synovial Fluid (SF) investigation should be a routine procedure for practitioners who perform arthrocentesis [1]. The most important reason to perform arthrocentesis is to check for joint infection, crystal deposition, autoimmunity, trauma or malignancy, as timely identification, treatment of a patient with septic arthritis are of paramount importance to a favorable clinical outcome. Also, arthrocentesis is indicated to gain diagnostic information through synovial fluid analysis in the case of a monoarticular or polyarticular arthropathy of unclear etiology presenting with joint pain and swelling [1]. Due to quality of relevant information obtained, the synovial fluid analysis has been referred as a “liquid biopsy” of the joint [2]. Proper SF investigation may decrease the number and cost of SF laboratory tests, shorten the time of recommended treatment initiation, and increase the odds of favorable prognosis [1,3].

shorten the time of recommended treatment initiation, and increase the odds of favorable prognosis [1, 3].

As the most important indication for SF investigation is infection identification or
exclusion, Gram stain and culture, total leukocyte count and differential are compulsory though not sufficient [4]. Besides infection, crystal deposition may also cause high grade joint inflammation [1]. In this setting SF crystal investigation is especially valuable in differential diagnosis of crystal deposition arthritis as the gouty arthritis is well controlled by uratelowering treatment while pyrophosphate or hydroxyapatite related arthritis requires symptomatic approach.
Thus, every obtained SF sample should be evaluated for the presence of pathological crystals. Polarized microscopy is recommended as standard diagnostic method of crystals detection. This statement may decrease general practitioners motivation to evaluate SF in absence of polarized microscope. Meanwhile, the ordinary light microscope, used by trained observers, is a quite sensitive tool for detecting the presence of crystals (either monosodium urate (MSU) or calcium pyrophosphate dihydrate (CPPD) in synovial fluid [2]. Two series of studies comparing the diagnostic significance of plain light and polarization microscopy demonstrated 96.2 to 100% sensitivity and 100% to 97.1% specificity of crystal identification by light microscopy [pascual]. Thus, if polarized microscopy is not available, the synovial fluid should be investigated using a conventional light microscope.
If MSU crystals cannot be detected initially, the slide should be allowed to dry and reexamined 3 hours or more later. Besides, CPPD crystals have been reported to be only weakly birifringent and are probably better identified by non-polarized light and characterized by morphology than by their polarization properties [2,3]. Also polarized microscopy may even cause misdiagnosis. So, negative polarized light microscopy synovial fluid examination report, does not exclude the presence of small numbers of MSUM or CPPD crystals.
Main crystals detection and identification issues may be related to several following factors:  MSU crystals are clearly visible in plain light, they are needleshaped, their size ranging from very small to about twice the diameter of a neutrophilic leukocyte. Tofus contents microscopy may be useful for learning how to identify these crystals. To do this, a white crumb-like material is placed on a slide and examined as a native preparation (Figure 1).    Thus, MSU crystals can be detected during routine SF microscopy and shorten the time between symptoms onset and diagnosis verification. CPPD crystals are more difficult to identify, since the threshold for their detection is a concentration of about 10-100MK/ml, while in natural conditions the concentration of crystals can be only 2-3MK/ml [7]. CPPD crystals size can range from 0.4 to 20

Calcium pyrophosphate dihydrate crystals
microns, most of them being less than 1 microns. This is exactly the threshold that allows them to be detected using non-polarized light microscopy [8]. CPPD identification can be performed by radiogram in advanced cases of the disease. We suggest that CPPD identification training should be performed using SF smears of patients with a previously established diagnosis of CPPD deposition disease.
CPPD crystals are polymorphic and can range from diamondshaped to brick-shaped ( Figure 6), as well as needle-shaped crystals, difficult to differentiate from MSU ( Figure 7). CPPD crystals are often located intracellularly (usually looking as small squares or needles) [9], even in the absence of inflammation (Figure 8).
It should be emphasized that, unlike MSU, CPPD microcrystalline arthropathy does not exclude septic arthritis [10], so the detection of CPPD crystals in the SF does not exclude the need for gram staining and bacteriological research. Azur-eosin staining also helps to identify CPPD crystals in SF (Figure 9).

Microscopic patterns of intra-articularly administered substances
Not rarely SF investigation reveals artifacts of administered substances, such as betamethasone or hydrocortisone suspension and lidocaine (Figure 10 & 11) which are visually like CPPD crystals.
Therefore, it is extremely important to follow the arthrocentesis procedure and carefully collect the patient's medical history.
Hyaluronic acid preparations are not detected by light microscopy and do not produce artifacts.