Annals of the Rheumatic Diseases, 1984, 43, 624-627

Transmission electron microscopic identification ofsilicon-containing particles in synovial fluid: potentialconfusion with calcium pyrophosphate dihydrate andapatite crystalsTHOMAS BARDIN,' H. RALPH SCHUMACHER,2 JULIETTE LANSAMAN,'SUSAN ROTHFUSS,2 AND ANTOINE DRYLL'

From the 'Clinique Rhumatologique de l'H6pital Lariboisiere, Paris, and the 2Veterans AdministrationMedical Center and University of Pennsylvania, Philadelphia, USA

SUMMARY Silicon-containing particles were identified by transmission electron microscopy(TEM) in thin sections of two synovial fluids, which also contained calcium pyrophosphatedihydrate (CPPD) crystals, aspirated during acute attacks of pseudogout. Such particles, whichare interpreted as probably being artefacts from glassware, were electron dense and similar inappearance to some CPPD or hydroxyapatite crystals.

Transmission electron microscopy (TEM) is, whenavailable, a useful technique for studying crystalrelated arthropathies. It can show crystals too smallto be seen by light microscopy -4 and allows furtherstudy of crystal-cell interaction. However, it can alsodisplay various artefacts, which may be confusing.5We report here the TEM observation of siliconcontaining particles which were found in synovialfluid preparations. These particles might have beenconfused with calcium pyrophosphate dihydrate(CPPD) or other crystals. We believe they wereartefacts from glassware.

Materials and methods

Synovial fluids from two patients were studied. Theyboth contained crystals suggestive of CPPD bycompensated polarising light microscopy and wereprocessed for TEM as part of a study of CPPDdeposition disease. No other unidentified particleswere noted on initial examination of these fluids aswet preparations.

Patient 1 was an 83-year-old woman who had hadseveral episodes of acute arthritis of her knees.Chondrocalcinosis of the knees, ankles, and shoul-ders was seen on x-rays. The fluid included in thisreport was aspirated during an acute knee arthritis.

Accepted for publication 23 February 1984.Correspondence to T. Bardin, MD, Clinique Rhumatologique del'H6pital Lariboisiere, 6 rue Guy Patin, 75010 Paris, France.

It contained 208 x 109 white cells/l. Patient 2 was a76-year-old female who had repeated attacks ofpseudogout of her left ankle without any radio-logical chondrocalcinosis. Synovial fluid included inthis report was aspirated during one of these attacks.It contained only 0-02 x 109 white cells/l. Neither ofthe patients had any underlying disease known to beassociated with CPPD deposition. Associated dis-eases included senile dementia in case 1 and renalphosphate wasting with normal serum parathor-mone (PTH) level in case 2.The two synovial fluids were aspirated with

disposable plastic syringes without the use of sterilegloves, then collected in glass vials containing glassballs which were mixed with the fluids to preventcoagulation before standard analysis. For electronmicroscopy the fluids were then transferred to glasstubes and centrifuged. Pellets were quickly placed in5% glutaraldehyde for half an hour at 4°C, thenminced in 1 x 1 mm pieces. Pieces were fixed in2-5% glutaraldehyde at 4°C for two hours, washedin cacodylate buffer, postfixed in 2% osmiumtetroxide, dehydrated in graded series of alcohol,and embedded in Spurr medium. Thin sections werecut with a diamond knife and observed in a ZeissEM 20 transmission electron microscope. X-raydispersive microanalysis was performed on un-stained carbon-coated thin sections in the sameTEM operated at 60 kV, with an Si (Li) x-raydetector interfaced with a multichannel analyser anda computer system.

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TEM identification of silicon-containing particles 625

Borosilicate and limeglass pipette fragments wereground and also embedded in Spurr medium. Thinsections of these materials were studied by TEM in asimilar way to the two synovial fluids.

Results

CPPD crystals were seen in both synovial fluids. ByTEM they appeared as electron dense rectangles of

Fig. 1 Dense quadrangularsilicon-containing particlewhich is located either insidethe cell or in an iAvagination.(Unstained thinsection, x 16 270). X-raymicroanalysis showed thisparticle to contain silicon andcalcium (insert).

Fig. 2 Dense silicon-containing particles

00 ~~misleadingly similar to CPPDcrystals. (x 11 110). Theseparticles also containedcalcium, chlorine, and sulphur(insert).

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626 Bardin, Schumacher, Lansaman, Rothfuss, Dryll

varying length, which became foamy during expo-sure to the electron beam. X-ray microanalysisshowed that they contained calcium and phosphoruswith aCa/P ratio approximating to one. Other electrondense particles were found to contain silicon (Fig.1). Their morphology was often very suggestive ofCPPD crystals except that they did not becomeincreasingly foamy under the electron beam. Thinsections of some silicon-containing particles didreveal a fine pitting with uniform holes that did notchange with beam exposure (Fig. 2). Silicon-containing particles could also appear as morefragmented masses sometimes divided into smallspicules (Fig. 3). Some such spicules could also bepotentially confused with apatite. X-ray micro-analysis showed that these particles often containedsmall amounts of calcium, and more rarely ofsulphur and chlorine, in addition to silicon. CPPDcrystals were both intra- and- extracellular.Silicon-containing particles were generally notfound inside cells. One silicon-containing fragmentpossibly related to a cell was seen in one unstainedsection (Fig. 1).TEM studies of the pipette fragments showed that

the glass debris obtained were electron dense andcontained silicon. Varying amounts of calcium inaddition to silicon were found in limeglass pipettesamples.

Discussion

This study has identified silicon-containing particlesin thin sections obtained from two synovial fluids.The morphology of these particles was diverse.Before microanalysis some of them were mistakenfor crystals and in particular for dense CPPDcrystals. However, the silicon-containing particlesdid not become foamy during the electron beamexposure as did CPPD crystals. An attentive ob-server could thus differentiate the two materials,though CPPD crystals are not the only crystal typeto have such a similar dense ultrastructure. Oxalatecrystals are known to have a close TEM resem-blance to CPPD,6 so that microprobe analysis orother analytical techniques7 are required fordefinite crystal identification.The finding of silicon in synovial fluids raises the

question of its origin and possible pathogenic role.Silica particles can absorb proteins and are knownto interact with macrophages. They can kill by a'perforation from within' mechanism9 1() and arebelieved to cause pulmonary lesions in silicosis.However, silica particles have never been heldresponsible for any otherwise unexplained arthritis.In our patients we have no evidence that thesilicon-containing particles are responsible for theirjoint inflammation. In both cases CPPD crystals

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Fig. 3 Silicon-containing particles fragmented into spicules which can be potentially confused with apatite crystals. Theseparticles did not contain any detectable element other than silicon. (x 6750).

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TEM identification of silicon-containing particles 627

were also present and could account for the acutearthritis. In one fluid a silicon-containing particlewas possibly within a cell, but phagocytosis canoccur in vitro as well as in vivo. Silica-containingparticles can break off silicone joint prostheses andappear in joint tissues,1" but in neither of ourpatients was the presence of silicon in a joint easilyexplainable. Neither patient had a history of anyobvious source of silica exposure or intake.2 On theother hand several steps in the processing of thesynovial fluid could explain the presence of silicon inthe final preparation. Disposable syringes were usedto aspirate synovial fluids. They contain a siliconcompound (polymethylsiloxane), serving as alubricant,'3 which could account for minute amountsof silicon in the fluids. Moreover, synovial fluidswere collected on glass balls to prevent clotting, andglassware was used during the preparation for TEM.Pipettes containing limeglass were used at severalsteps of the processing and could account for thefrequent association of calcium with the silicon-containing particles. Therefore it seems highly prob-able that the variously shaped silicon-containingparticles were artefacts from glassware. Investiga-tors should be aware that such artefacts can have anappearance on transmission electron microscopythat can mislead one into suspecting CPPD orapatite crystals.

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