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Diagnostic potential of rapid electron microscopic analysis of joint effusions.

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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,
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.
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
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).
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
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).
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).
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,
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,
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,
11. S p u n AR: A low-viscosity epon resin embedding medium for electron microscopy. J Ultrastruct Res 26:31-43,
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,
The authors are grateful for the technical assistance
of Mrs. Susan Rothfuss, Miss Marie Sieck, and Miss Gilda
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potential, rapid, effusion, microscopy, joint, electro, analysis, diagnostika
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