Synovial Biopsy, Synovium, Synovial Fluid & Arthrocentesis
Synovial Fluid Analysis. Figures related to the following text: Photo/Slide Viewer
Current concepts of gout and CPPD deposition disease began with the microscopic identification of crystals in synovial fluid by Hollander and McCarty in 1961-1962 (1) and microscopic examination continues to be the principal method for crystal identification. Gout in fact is defined by the presence of monosodium urate crystals. CPPD crystals can be seen in a variety of settings, may be a result of osteoarthritis or other joint diseases and do not as clearly define a disease. Other crystals have now also been identified in joint fluids. These have a variety of implications.
Synovial fluid analysis for crystals is generally considered the gold standard. It can change clinically suspected diagnoses and can change treatment (2) but it does have limitations. Reliability of identification depends on training and experience of examiners and on the quality and proper maintenance of the microscope. Several studies have reported wide discrepancies of results on aliquots of fluid sent to different laboratories (3).
Evidence for the presence of crystals can begin to accumulate with the initial gross examination of the fluid aspirated (4). Some gouty fluids will have visible white chunks (Fig. 1)that are virtually diagnostic and can be confirmed quickly on microscopic examination to be masses of MSU crystals from a tophaceous deposit in the joint. Less commonly, fluids aspirated from joints or bursas can be milky white liquids or pastes. These should raise suspicion for either gout or apatite disease. CPPD crystals rarely if ever produce such white effusions. Massive cholesterol crystal laden fluids as occur in some chronically swollen shoulders or olecranon bursas may glisten with a gold paint appearance.
The gross appearance of the fluid may also give important information about the host response to crystals. Totally clear, almost colorless, viscous fluids may still contain MSU or CPPD crystals but suggest that there is little or no inflammatory response to the crystals. During acute attacks of gout or pseudogout effusions are cloudy because of the leukocyte response. Microscopic examination of a wet drop, differentials on a stained smear and leukocyte counts are commonly used to further characterize the response.
Effusions occasionally are bloody with any of the crystal induced diseases but this may be especially common in the apatite associated destructive disease in the Milwaukee shoulder syndrome.
Examination of a Wet Drop Preparation
The standard approach to crystal identification still follows the preparation of a single wet drop of joint fluid as utilized by Hollander and McCarty (1). A single drop tends to have less distracting movement of the fluid. If few cells are present effort may be needed to focus on the proper plane. Look with 10X or 40X objectives for cells, other debris, fat etc. It may help to start at the margin of the cover slip where cells and crystals may clump.
The authors recommend examining the preparation first using regular light before proceeding to the generally definitive use of compensated polarized light. This allows a quick impression about the cellular content of the fluid, identification of other possibly important features such as fat droplets, fibrils and cartilage or synovial fragments, and may provide the first clue to the presence of apatite (basic calcium phosphate) crystal clumps. These latter can appear as shiny, round or irregular 3-15 μ chunks (Fig 2) (5). MSU and CPPD crystals can be seen and their nature suggested by their shapes and sizes. MSU crystals may be long needles up to 30 μ but also 3-10 μ rods. CPPD can also be rodlike but more characteristically are square, rhomboid or some may be irregular and are mostly 3-7 μ . Since CPPD are often only weakly or virtually non-birefringent they may actually be best seen on this regular light examination. Phase contrast or adjusting the condenser may produce a phase effect and can better delineate the CPPD crystals. Oil immersion may help identify small crystals. Especially when there are few cells, crystals may be best seen in clumps of tissue fibrils or tissue fragments.
Concentration of Specimens
If crystals are not seen on several minutes search, time can be saved and accuracy assured by also concentrating one aliquot of fluid by centrifugation or use of a cytocentrifuge (6). Crystals may be missed on initial examination of fluids for a variety of reasons beside paucity of crystals (7). Occasionally all crystals may be very small. The effusion aspirated may be from an adjacent sympathetic effusion not from the actual site of crystal induced inflammation.
Examination of Stained Smears or Pellets
Dried smear preparations of inflammatory effusions can be stained and examined for crystals. Such specimens can be saved for later re-examination without concern for development of artifacts. Wright’s stain or Diff Quick preparations (8,9) have been reported to show either CPPD or MSU crystals which are especially easily characterized in cells (Fig.3). Gram stains prepared in microbiology laboratories have also shown crystals in specimens with or without stained bacteria.
Staining of Wet Preparations
The best studied but still not widely used technique for crystal identification is alizarin red S staining for apatites and other calcium containing crystals. This procedure described by Paul et al (10) stains apatite containing clumps as bright red, round, occasionally Chinese coin-like or irregular 3 – 15 μ bodies (Fig. 4). CPPD and oxalate (Fig. 5) will also stain red but retain their typical shapes and stain more slowly. Urate crystals have been stained with De Golantha silver stain (11) or methylene blue but any clinical value of these has not been tested. A preparation called Testsimplets (Boehringer-Ingelheim) which has a dry stain on the slide and stains viable cells in fluid applied as a wet drop defines cells well and does not dissolve crystals (12).
Compensated Polarized Light
Birefringent crystals such as MSU and CPPD can be highlighted and further characterized by use of polarized light which is best performed with commercial polarizing microscopes (13 ). In such microscopes a polarizing plate is placed above the light source. This orients the light into many parallel planes. A second similar polarizer (called the analyzer) above the specimen can be rotated 90° to these parallel planes of light. When this is done no light passes through and the field appears dark. If the specimen contains crystals these will cause the direction of the light planes to deviate so that bright white light will now pass through the analyzer and the examiner will see a white crystal shape against the dark background.
The technique now widely used to help distinguish MSU and CPPD crystals involves use of a first order red plate or compensator inserted between the polarizer and analyzer. This turns the background red. Crystals appear blue or yellow and their orientation can be noted in relation to the axis of slow vibration of the compensator. This is usually marked by a line or arrow. The long axis of the crystal being examined is then aligned with the orienting arrow. If the crystal is blue in this position it is said to have positive elongation. This is characteristic of most CPPD crystals (Fig. 6) (14). Crystals yellow parallel to the axis of slow vibration are termed to have negative elongation as is typical for MSU crystals (Fig. 7). If crystals are rotated or other crystals appear in the field their color will be opposite when perpendicular to the line of orientation. That is a MSU crystal will be blue when perpendicular to the axis of slow vibration.
Several other features can be noted. Blue or yellow birefringence will often disappear when rotated to midway between parallel and perpendicular to the orienting axis. This is termed extinction. MSU crystals are virtually always very bright. CPPD crystals can be bright but often are much less birefringent and occasionally show no birefringence with polarized light (15).
Less common crystals can also be seen with compensated polarized light. These and a variety of artifacts need to be distinguished from the common pathogenetic crystals (16).
A variety of techniques not considered part of a practical synovial fluid analysis should also be mentioned as they may be used in research or called upon in rare situations to identify a puzzling finding in a synovial fluid.
Electron microscopy can identify very small crystals by morphology (Fig. 8) (17) and can allow confirmation by electron diffraction elemental analysis (5) (Fig. 9). X-ray diffraction or Fourier transform infra red analysis are the most definitive methods for crystal identification but require more crystals than are often available. A technique using 14Carbon labelled ethane-1-hydroxy-1 diphosphonate (EHDP) can detect small amounts of apatite but is not generally available (13,18). A recent report describes dissolution of urate crystals in synovial fluid with their measurement of soluble urate as a possible way to quantify urates (19).
Other Less Common Crystals and Artifacts
A rare example of suspected crystal associated arthritis will turn out to be related to or associated with a variety of less common crystals (16).
Oxalate crystals can complicate renal failure and appear as double pyramids or envelopes (20). Depot corticosteroids can vary in shape and size but are generally very brightly birefringent (21). These can cause an iatrogenic acute inflammation after injection in occasional patients (22). Liquid lipid crystals can appear like maltese crosses or beach balls and can be phagocytized and associated with acute inflammation (23) (Fig.10). The broad notched plates typical of cholesterol crystals are generally not phagocytized or phlogistic. Proteins such as cryoglobulins can form crystals; these and Charcot-Leyden crystals from eosinophilic exudates (Fig. 11)have been associated with arthritis (16).
Crystal-like artifacts include glass fragments, lipid crystals developing from cell breakdown, starch from gloves, oxalate, EDTA or lithium anticoagulants, hemoglobin breakdown products, drying artifacts, dust and lens paper fibrils (24).
Identification of synovial fluid crystals assumes ability to successfully obtain synovial fluid. Patients may initially decline arthrocentesis but may be more willing if made aware that diagnosis can be wrong up to 20% of the time without proof of crystal presence and identity (2). Large bulging effusions at knees may be relatively easy to aspirate but accurate and successful needle placement in knees may be achieved less often than many suspect. Confirmation by X-ray contrast injection has been used to study accuracy of injections (25,26). The superolateral route of entry into the suprapatellar pouch was most successful, medial retropatellar aspiration second best and aspiration of the flexed knee medial or lateral to the patellar tendon least reliable. Shoulder arthrocentesis is less often successful. First MTP joints (and others) can be aspirated even without a detectable effusion (27) but no studies document rates of success. Ultrasound has been suggested to improve success with small effusions (28). Routes for aspiration of various other joints such as the shoulder have been described (29).
No generally available system for quality control has been established for crystal identification. Ability to identify crystals in text books or lecture slides has not been confirmed to identify ability to identify the crystals in a given laboratory. A system to distribute embedded samples of crystals for use by individuals on their own microscopes has been proposed (30) but does not reproduce the clinical experience of the preparation techniques or totally reproduce the appearance of clinical specimens.
1. McCarty DJ, Hollander JL. Identification of urate crystals in gouty synovial fluid. Ann Int Med, 1961; 54:452-.
2. Eisenberg JM, Schumacher HR, Davidson PK, Kauffman L. Usefulness of synovial fluid analysis in the evaluation of joint effusions. Use of threshold analysis and likelihood ratios to assess a diagnostic test. Arch Int Med 1984; 144:715-719.
3. Schumacher HR, Sieck MS, Rothfuss S, et al: Reproducibility of synovial fluid analyses: a study among four laboratories. Arthritis Rheum 1986, 29:770-774.
4. Schumacher HR, Reginato AJ: Atlas of Synovial Fluid Analysis and Crystal Identification. Philadelphia: Lea & Febiger; 1991.
5. Schumacher HR, Somlyo AP, Tse R, Maurer K. Apatite crystal associated arthritis. Ann Int Med 1977; 87:411-416.
6. Moreno MJ, Clayburne G, Schumacher HR: Processing of non-inflammatory synovial fluids with hyaluronidase for cytospin preparations impromes the accuracy of differential counts. Diagn Cytopathol 2000, 22:256-258.
7. Schumacher HR, Jimenez SA, Gibson T, et al.: Acute gouty arthritis without urate crystals identified on initial examination of synovial fluid: report of 9 patients. Arthritis Rheum 1975, 18:603-612.
8. Petrocelli A, Wong AL, Swezey RI: Identification of pathologic synovial fluid crystals on Gram stains. J Clin Rheum 1998, 4:103-105.
9. Selvi E, Manganelli S, Catenaccio M: Diff Quik staining method for detection and identification of monosodium urate and calcium pyrophosphate crystals in synovial fluid. Ann Rheum Dis 2001, 60:194-198.
10. Paul H, Reginato AJ, Schumacher HR: Alizarin red S stainlng as a screening test to detect calcium compounds in synovial fluid. Arthritis Rheum 1983, 26:191-200.
11. de Galantha E: Techniques for preservation and microscopic demonstration of nodules in gout. Am J Clin Pathol 1935, 5:165-166.
12. Louthrenoo W, Sieck M, Clayburne G, et al.: Supravital staining of cells in noninflammatory synovial fluids: Analysis of the effect of crystals on cell populations. JRheumatol 1991, 18:409-413.
13. Gatter RA, Schumacher HR. A practical handbook of joint fluid analysis. Lea and Febiger, Philadelphia 1991 2 nd Ed. 122 p.
14. Kohn NN, Hughes RE, McCarty DJ, Farres JS. The significance of calcium phosphate crystals in the synovial fluid of arthritic patients: The “pseudogout syndrome”. II. Identification of crystals. Ann Int Med 1962; S6:738-
15. Ivorra J, Rosas J, Pascual E: Most calcium pyrophosphate crystals appear as non-birefrigent. Ann Rheum Dis 1999, 58:582-584
16. Schumacher HR. Other crystals. In Klippel JH, Dieppe PA (Eds) Rheumatology , Mosby, St. Louis 1994, 7-15.1 – 7.15.4.
17. Cherlan V, Schumacher HR. Diagnostic potential of rapid electron microscopic analysis of joint effusions. Arth Rheum 1982; 25:98-100.
18. Halverson PB, McCarty DJ. Identification of hydroxyapatite crystals in synovial fluid. Arth Rheum, 1979; 22:389- .
19. Tiliakos AN, Tiliakos NA: Total joint fluid urate in gout. J Clin Rheum 2004; 10:250-251.
20. Hoffman GS, Schumacher HR, Paul H et al.: Calcium oxalate microcrystalline associated arthritis in end stage renal disease. Ann Intern Med 1982;97:36-42.
21. Kahn CB, Hollander JL, Schumacher HR. Corticosteroid crystals in synovial fluid. JAMA 1970; 211:807-809.
22. Gordon GV, Schumacher HR. Electron microscopic study of depot corticosteroid crystals with clinical studies after intra-articular injections. J Rheumatol 1979;6:7-14.
23. Reginato AJ, Schumacher HR, Allan DA, Rabinowitz JL. Acute monoarthritis associated with lipid liquid crystals. Ann Rheum Dis. 1985; 44:537-543.
24. Chen LX, Clayburne G, Schumacher HR. Update on identification of pathogenic crystals in joint fluid. Current Rheum Reports 2004; 6:217-220.
25. Schumacher HR: Aspiration and injection therapies for joints. Arthritis Care Res 2003, 49:413-420.
26. Jackson DW, Evans NA, Thomas BM. Accuracy of needle placement into the intraarticular space of the knee. JBJS 2002; 84A:1522-1526.
27. Aqudelo CA, Weinberger A, Schumacher HR, Turner RA, Molina J. Definitive diagnosis of gout by identification of urate crystals in asymptomatic metatarsophalangeal joints. Arth Rheum 1979; 22:559-560.
28. Grassi E, Farina A, Fillippucci E, Cervini C, Sonographically guided procedures in rheumatology. Semin Artls Rheumatol 2001, 30:347-353.
29. Schumacher HR. Arthrocentesis of the shoulder. Hospital Med 1997; ___:57-60.
30. Schumacher HR, Sieck M, Clayburne G: Development and evaluation of a method for preservation of synovial fluid wet preparations for quality control testing of crystal identification. J Rheumatol 1990, 17:1369-1374.
More References Related to Synovial Fluid Analysis
Schumacher HR: Analyzing synovial fluid. A useful diagnostic aid for the practitioner. Modern Medicine 45:58-63, 1977.
Paul H, Reginato AJ, Schumacher HR: Alizarin red S staining as a screening test to detect calcium compounds in synovial fluid. Arthritis Rheum 26:191-200, 1983.
Schumacher HR, Sinkinson CA , Weiss JJ: Guidelines for obtaining and analyzing synovial fluid. ER Reports 4:37-43, 1983.
Schumacher HR: Synovial fluid analysis. Orthopaedic Review 5:85-92, 1984.
Eisenberg JM, Schumacher HR, Davidson PK , Kauffman L: Usefulness of synovial fluid analysis in the evaluation of joint effusions. Use of threshold analysis and likelihood ratios to assess a diagnostic test. Arch Int Med 144:715-719, 1984.
Schumacher HR, Cherian PV: Transmission electron microscopic studies on articular calcium crystals and associated protein coatings. Scanning Electron Microsocopy II: 964-968, 1984.
Schumacher HR, Sieck MS, Rothfuss S, Clayburne GM, Baumgarten DF, Mochan BS, Kant JA: Reproducibility of synovial fluid analyses. A study among four laboratories. Arthritis Rheum 29:770-774, 1986.
Villanueva TG, Schumacher HR: Cytologic examination of synovial fluid. Diag Cytopath 3:141-147, 1987.
Kerolus G, Clayburne G, Schumacher HR. Is it mandatory to examine synovial fluids promptly after arthrocentesis? Arth Rheum 32:271-278, 1989.
Schumacher HR, Sieck M, Clayburne G: Development and evaluation of a method for preservation of synovial fluid wet preparations for quality control testing of crystal identification. J Rheumatol 17:1369-74, 1990.
Louthrenoo W, Sieck M, Clayburne G, Rothfuss S and Schumacher HR: Supravital staining of cells in noninflammatory synovial fluids:Analysis of the effect of crystals on cell populations. J Rheumatol 18:409-413, 1991.
Clayburne G, Baker DG, Schumacher HR: Estimated synovial fluid leukocyte numbers on wet drop preparations as a potential substitute for actual leukocyte counts. J Rheumatol 19:60-62, 1992.
Ferrari AJL, VanLinthoudt D, Schumacher HR: Evaluation of synovial fluids for crystals. Rheumatol Rev 1:193-203, 1992.
Schumacher HR: Is examining synovial fluid useful in primary care settings? Contemporary Int Med 8:22-27, 1996.
Moreno MJ, Clayburne G, Schumacher HR: Processing of noninflammatory synovial fluids with hyaluronidase for cytospin preparations improves the accuracy of differential counts. Diagnostic Cytopathology 22:256-8, 2000.
Chen LX, Clayburne G, Schumacher HR: Update on identification of pathogenic crystals in joint fluid. Cur Rheum Rep 6:217-220, 2004.
Schumacher HR: Office analysis of synovial fluid; Outpatient procedure. Hospital Medicine 34:38-41,1998.
Dai L, Pessler F, Chen LX, Clayburne G, Schumacher HR: Detection and initial characterization of synovial lining fragments in synovial fluid. Rheumatology ( Oxford ), 2005 [epub ahead of print] .
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