Diagnostic potential of rapid electron microscopic analysis of joint effusions.код для вставкиСкачать
98 BRIEF REPORT DIAGNOSTIC POTENTIAL OF RAPID ELECTRON MICROSCOPIC ANALYSIS OF JOINT EFFUSIONS P. VARGHESE CHERIAN and H. RALPH SCHUMACHER The use of electron microscopy (EM) in clinical diagnosis of various diseases has been limited in part because of the lengthy procedures involved. In joint diseases EM analysis would be very helpful in identifying small crystals which can cause arthritis. Monosodium urate and calcium pyrophosphate crystals are at least occasionally too small to be seen by light microscopy (1-3). By light microscopy, apatite crystal clumps may infrequently appear as “shiny coins,” but these tiny crystals are most often identified by EM (45 ) . EM can also help identify larger crystals, when absolute identification is needed. X-ray diffraction can also do this but the amount of material available is often insufficient. Compensated polarized light is almost diagnostic for sodium urate and calcium pyrophosphate crystals, but other crystals such as depot corticosteroids (6) can mimic their shape and birefringence. EM allows elemental analysis (4) and electron diffraction (7) in addition to morphologic description and can thus definitively identify crystals. The conventional manner of preparing biological samples for EM takes 3-5 days before sections can ______ From the School of Medicine, University of Pennsylvania, and the Arthritis-Immunology Center, Veterans Administration Medical Center, Philadelphia, PA. Supported in part by grants from the Kroc Foundation, Barsumian Fund, and McCabe Foundation. P. Varghese Cherian, PhD: Research Associate, Department of Medicine, University of Pennsylvania, School of Medicine, and the Veterans Administration Medical Center, Philadelphia, PA; H. Ralph Schumacher, MD: Professor of Medicine, University of Pennsylvania School of Medicine, and Director, Arthritis-lmmunology Center, Veterans Administration Medical Center, Philadelphia, PA. Address reprint requests to H. Ralph Schumacher, MD, Director, Arthritis-Immunology Center, Veterans Administration Medical Center, University and Woodland Avenues, Philadelphia, PA 19104. Submitted for publication January 23, 1981; accepted in revised form May 22, 1981. Arthritis and Rheumatism, Vol. 25, No. 1 (January 1982) be examined. Much of this time is consumed during the washing procedure, infiltration, and curing of the embedding medium. Several methods to shorten the EM procedure by reducing the washing and centrifugation time have been reported (8-9). Recently, Rittenburg et a1 (10) used polycarbonate membrane filters to shorten the duration of most steps for processing microorganisms for EM. The present study was designed to determine whether synovial fluid components could be processed rapidly enough to provide information about crystals or other fluid components the same day that fluid is aspirated for diagnosis and management. Our technique provided a rapid (3 hours, 40 minutes) and efficient method for preparing synovia1 fluid cells and crystals which retain well-preserved morphology, suggesting that EM could be useful in the rapid clinical diagnosis of crystal deposition disease and possibly in other diseases. MATERIALS AND METHODS Two milliliters of joint effusion received from a patient (MS) with known pseudogout and apatite crystal deposition disease and a 2.0-ml sample from another patient (MZ) containing only equivocal crystals by light microscopic analysis were washed in phosphate buffered saline with 2 changes in a 10-minute period and then centrifuged for 10 minutes at 2,500g. The supernatants were discarded and the pellets were fixed in 3 ml of 4% glutaraldehyde in O.IM cacodylate buffer, pH 7.4, for 30 minutes at room temperature. The fixative solution was removed and the pellets were washed with 5.0 ml of 0. IM cacodylate buffer for 2 minutes. They were immediately postfixed in 2 ml of 2% osmium tetroxide in 0.1M cacodylate buffer for 25 minutes at 4°C. After being rinsed with distilled water, the pellets were stained en bloc with 2% aqueous BRIEF REPORTS Figure 1. Electron micrograph of synovial fluid cell of patient MS with apatite crystals (ap) inside phagocytic vacuoles (pv). The endoplasmic reticulum (rer). mitochondria (m)with cristae. and nucleus (n) are clearly visible ( x 30.000). 99 chondria with cristae, nuclei, and other cellular components (Figures I , 2). Hydroxyapatite and calcium pyrophosphate crystals were observed in both synovia1 fluid preparations. Several of the synovial fluid cells had apatite and calcium pyrophosphate crystals in phagocytic vacuoles (Figures 1 , 2). Needle-shaped 725 nm hydroxyapatite crystals (4) were present individually (Figure 1) and in clusters. with the crystals lying in a grainy matrix in phagocytic vacuoles and outside the cell. Calcium pyrophosphate crystals were characteristically foamy in appearance (3) (Figure 2); they were denser and larger than hydroxyapatites. They tended to break apart during cutting of thin sections and further dislodge and deteriorate under the electron beam. Both types of crystals were confirmed as containing calcium and phosphorus by energy dispersive elemental analysis (4). In the apatite crystals the ratio of calcium to phosphorus was as expected (41, approximately 1.6: I , while with the calcium pyrophosphate crystals the calcium-to-phosphorus ratio was 1:l. uranyl acetate for 5 minutes. The samples were dehydrated by being washed in 3 ml each of 50%, 7076, 95%, and 100% acetone; the samples were left for 1 minute in each solution and changed twice in the 100% acetone. The pellets were next soaked in a I : 1 mixture of acetone and Spurr embedding medium (Polysciences, Warrington. PA) for 30 minutes and then in a 1:2 mixture for 15 minutes. The pellets were then infiltrated with fresh embedding medium, with 2 changes of 10 minutes each, cut into very small pieces, and embedded in flat embedding molds or polethylene capsules. Specimens were cured at 99°C for 1 hour. Blocks were sectioned 7 minutes after removal from the oven with a diamond knife and examined with a Zeiss 10 electron microscope at 60 kV. Energy dispersive elemental analysis of all crystals identified was performed on the same thin sections using a Kevex Si (Li) x-ray detector interphased with a multichannel analyzer and computer. Crystals were analyzed using spot sizes of 0.5-2 pm diameter and 120-200 second analysis times (13). RESULTS Synovial fluid cells and crystals retained wellpreserved ultrastructural details. Cells displayed intact cell membrane, rough endoplasmic reticulum. mito- Figure 2. Electron micrograph of synovial fluid cell of patient MS with calcium pyrophosphate (CPPD) crystals within a phagocytic vacuole (pv) ( x 30.000). BRIEF REPORTS 100 DISCUSSION REFERENCES This method allowed cells and various crystals in the synovial fluid to be processed rapidly with milder preparatory steps than the conventional methods. Synovial fluid components formed a pellet on centrifugation and became firm during fixation. It was thus possible to wash with various solutions without disrupting the pellet. We also experimented with polycarbonate filter membranes forjoint fluids, as has been suggested for microorganisms (lo), but we found that centrifugation of the sample and processing the resulting pellet were more sensitive. If one were particularly looking for crystals, postfixation in osmium tetroxide and staining en bloc with uranyl acetate could be eliminated because crystals would have sufficient contrast in the absence of these compounds. This would reduce the time by one-half hour. Low-viscosity Spurr embedding medium allows better morphologic preservation of a variety of substances including minerals, rocks, and endosperm (1 1-12). The penetration quality of Spurr embedding medium may have accounted for the well preserved morphology of the synovial fluid cells and crystals. Monosodium urate and other watersoluble crystals were dissolved during preparation for transmission EM (1,2,14,15). Their characteristic outline remained in the thin section and could suggest the presence of some water-soluble crystal. This phenomenon occurred in our previous study (1) and that of Honig et a1 (2). Our results suggest the usefulness and efficiency of electron microscopy in establishing a definitive diagnosis in crystal deposition diseases. The limit of resolution of light and polarized light microscopy and the relatively low number of crystals in some cases may leave small sodium urate and calcium pyrophosphate crystals as well as apatite crystals undetected with only light microscopy (1,2). The significance of very small crystals is not definitely established in all effusions, although in some patients they have appeared to explain inflammation (1-2,4). Arbitrary exclusion of crystals by small size, however, also does not seem appropriate at this time. 1. Schumacher HR, Jimenez SA, Gibson T, Pascual E, Traycoff R, Dorwart BB, Reginato AJ: Acute gouty arthritis without urate crystals identified on initial examination of synovial fluid: report on nine patients. Arthritis Rheum 19:603-612, 1975 2. Honig S, Gorevic P, Hoffstein S, Weissmann G: Crystal deposition disease: diagnosis by electron microscopy. Am J Med 63:161-164, 1977 3. Schumacher HR: Ultrastructural findings in chondrocalcinosis and pseudogout. Arthritis Rheum 19:413-425, 1976 4. Schumacher HR, Somlyo AP, Tse RL, Maurer K: Arthritis associated with apatite crystals. Ann Intern Med 87:411-416, 1977 5. Dieppe PA, Crocker P, Huskisson EC, Willoughby DA: Apatite deposition disease: a new arthropathy. Lancet 11 ~266-269, 1976 6. McCarty DJ, Hogan JM: Inflammatory reaction after intrasynovial injection of microcrystalline adrenocorticosteroid esters. Arthritis Rheum 7:359-367, 1964 7. Pritzker KPH, Adams ME, Cheng P-T, Omar SA, Cruickshank B: Black synovium: a consequence of intraarticular gold therapy. Arthritis Rheum 23:49&504, 1980 8. Hayat MA: Basic Electron Microscopic Techniques. New York, Van Nostrand Reinhold, 1972, pp 2-4 9. Dalen H: A filtration technique for preparing cells in suspension for electron microscopy. J Microscopy 91:213-215, 1970 10. Rittenburg JH, Bayer RC, Gallagher ML, Leavitt DF: A rapid technique for preparing microorganisms for transmission electron microscopy. Stain Techno1 54:275-280, 1979 11. S p u n AR: A low-viscosity epon resin embedding medium for electron microscopy. J Ultrastruct Res 26:31-43, 1969 12. Spurr AR, Harris WM: Ultrastructure of chloroplasts and chromoplasts in Capsicum annum. I. Thylakoid membrane changes during fruit ripening. Am J Bot 55: 1205-1209, 1968 13. Schumacher HR, Miller JL, Ludivico C, Jessar RA: Erosive arthritis associated with apatite crystal deposition. Arthritis Rheum 24:31-37, 1981 14. Schumacher HR: Pathology of the synovial membrane in gout: light and electron microscopic studies: interpretation of crystals in electron micrographs. Arthritis Rheum 18:771-782, 1975 15. Gordon GV, Schumacher HR: Electron microscopic study of depot corticosteroid crystals with clinical studies after intra-articular injection. J Rheumatol 6:7-14, 1979 ACKNOWLEDGMENTS The authors are grateful for the technical assistance of Mrs. Susan Rothfuss, Miss Marie Sieck, and Miss Gilda Clayburne.