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The synovitis of pseudogoutelectron microscopic observations.

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The Synovitis of Pseudogout : Electron Microscopic
Observations
By H. RALPHSCHUMACHER,
JR.
Specimens of synovium were obtained by ParkerPearson needle biopsy0 from multiple areas of the
acutely inflamed knee joint of a 60-year-old woman
with classical pseudogout. The patient recalled two
previous episodes of monarticular acute inflammation (most recently in the same knee 2 years
earlier). She continued to have mild arthralgia of
the knee until the onset of acute arthritis 2 days
before biopsy. X-rays showed dense wafer-like
calcification of the menisci of both knees. The
synovial fluid was viscous, yellow and turbid and
produced a normal mucin clot. There were 26,900
WBC's/cu.mm. (98 per cent polymorphonuclears,
2 per cent lymphocytes) and 25,300 RBC's/cu. mm.
Culture was negative. Numerous rod-like to rhombic intra- and extra-cellular crystals were present
and with polarized light exhibited a weakly positive sign of birefringence. There was no evidence
to suggest other concomitant rheumatic disease
or primary cause for the chondrocalcinosis. Serum
uric acid, calcium, phosphorus and alkaline phos-
phatase were normal. A latex fixation test was
negative.
Specimens of synovial membrane and exudate
were prepared for both light and electron microscopy. Portions of tissue for the former were
6xed in either 4 per cent formol-saline or 95 per
cent ethyl alcohol and examined unstained and
after Wright's stain or hematoxylin and eosin.
For electron microscopy, the small specimens
of synovium were placed immediately in Karnovsky's formaldehyde-glutaraldehyde fixative diluted 1:l with 0.1 M cacodylate buffer at pH 7.4.7
The synovial fluid was centrifuged and the pellet
immersed in the same fixative. After 30 minutes'
fixation, the specimens of synovium and pellet of
exudate were diced into 1 x 1 mm. pieces and
replaced for a total of 4 hours, at room temperature, in the fixative solution.
Specimens were washed in 0.1 M sodium cacodylate buffer at 4 C overnight. They were then
post-fixed in cold Palade's osmium-veronals.@ for
2 hours, dehydrated in alcohol, placed in a 50 per
cent propylene oxide and Epon mixture for 2
hours, and then embedded in Epon 812. Thick
sections ( 1 ~ were
)
cut on an LKB-1 ultramicrotome.
with a glass knife, stained with toluidine blue and
examined for orientation. Thin sections were cut
with a diamond knife and stained with uranyl acetate and lead citrate and examined on an RCA
EMU-3F electron microscope under a 50 KV beam.
Nine blocks of synovial membrane and 3 blocks
of synovial fluid exudate were examined.
Synovium from a needle biopsy performed on
another patient with pseudogout was fixed in 95
per cent alcohol, in an attempt to prevent loss of
crystals in the usual water-based fixatives. A portion of this was later embedded as above for electron microscopy. Crystals obtained at operation
from a frosty-appearing exudate coating a portion
of the articular cartilage of a third patient, were
dried and then embedded and examined with the
electron microscope.
This work was supported by NIH Grant TI
AM 05076-11
H. RALPH SCWLIMACHER,
JR., M.D.: Research
Fellow, Department of Pathology, Haruard Medical School, Robert B . Brigham Hospital; Assistant
in Medicine, Peter Bent Brigham Hospital. Address
Requests for Reprints to: 206 Maloney Clinic Building Hospital of the University of Pennsyluania,
36th and Spruce Streets, Philadelphia, Pennsylzjania 19104.
S
changes in pseudogout
( chondrocalcinosis articularis ) have
been extensively studied, and the pathogenic role of phagocytosis of crystals by
polymorphonuclear leucocytes has been
emphasized.le2 The histopathology of the
associated synovitis has been described by
McCarty and his collaborators,lS2 de SBze
et al.,3 Currey et al.,4 and Kulka and Schumachers but the ultrastructure has not been
investigated. This electron microscopic
study was, therefore, initiated to further
characterize the synovial membrane alterations and assess their possible role in this
still incompletely understood disease.
YNOVIAL FLUID
MATERIALS
AND METHODS
426
ARTHRITISAND RHEUMATISM,VOL. 11, NO. 3 (JUNE 1968)
SYNOVlTIS OF PSEUDOGOUT
427
Fig. 1.-Synovial lining layer containing multiple clear clefts (C) from which
crystals have been dissolved. J = Joint space. LC = Nucleus of lining cell. CF =
Collagen fibers. Above LC, note apparent necrosis of a crystal laden cell. ~ 4 5 0 0 .
RESULTS
Light microscopy of the paraffin-embedded synovial membrane and thick sections
of the Epon-embedded tissue showed vascular congestion, proliferation of the synovial lining cells, and fibrosis. Surface exudate contained fibrin, neutrophils and
occasional enmeshed fragments of cartilage.
The synovium was focally infiltrated by
neutrophils, lesser numbers of lymphocytes,
other small mononuclear cells and plasma
cells.
Typical birefringent crystals of calcium
pyrophosphate were abundant in the exudate and also present, but less frequent, in
the supeAcial synovium. They were most
consistently seen on unstained sections or
with undiluted Wright's stain after 95 per
cent alcohol fixation, and could easily have
been missed on the hematoxylin and eosinstained slides.
On electron microscopy, clear rodlike,
rhombic, acicular or irregular spaces, presumably the sites from which crystals had
been dissolved by the water-based fixatives,
were found in the interstitium, in hyperplastic synovial lining cells (Figs. 1 and 2)
and in neutrophils and macrophages. Only
occasionally were intact crystals or clearly
distinguishable remnznts of crystals noted.
These almost invariably showed a lacelike
internal structure (Fig. 3) although a few
were uniformly electron-dense. Some intracellular crystal-shaped spaces and crystals
(hereafter both referred to as crystals)
were noted in lining cells with many dense
bodies and vacuoles. Many crystals were
distinctly membrane-bounded and appeared to be in phagosomes (Figs. 3 and 4 )
which sometimes also contained fibrin and
cellular debris. Only occasionally did dense
bodies (presumably lysosomes) surround
crystal containing phagosomes. Other crystals were found in cells rich in rough endo-
H. RALPH SCHUMACHER, JR.
428
Fig. S.--Synovial lining cell (probably intermediate type) with clear residual
clefts from crystal deposits (C), dense bodies (D), endoplasmic reticulum (ER)
and mitochondria (M). J = Joint space. F = Fibrin-like material. x 12,000.
plasmic reticulum with only rare dense
bodies and without identifiable phagosomes
(Fig. 5). A definite enclosing membrane
could not be seen around some crystals.
Fibrin, free cellular organelles, and degenerated nuclear material were noted on
the synovial membrane surface and in the
interstitium. Many of the synovial lining
cells with well-developed rough endoplasmic reticulum or deeper fibroblast-like cells
showed focal lipid deposits. Mast cells appeared to be normal. Inflammatory cells
were predominantly neutrophils but lymphocytes were also easily identified. No
crystals were seen in typical lymphocytes.
Degenerating neutrophils were seen within
macrophages (Fig. 6). These macrophages
also contained other phagocytized material,
including fibrin, lipid and various organelles.
The small blood vessels showed no evidence of thrombosis or necrosis of their
walls but a number of thin walled vessels
of 10-60p diameter did have definite gaps
between endothelial cells. Erythrocytes
were occasionally identified in the vascular
wall (Fig. 7 ) and neutrophils could be seen
in the perivascular region and apparently
emigrating through endothelial gaps. Fig.
8 shows a small vessel containing erythrocytes and neutrophils with a process of one
leucocyte apparently beginning to pass between two endothelial cells. Large num-
Fig. 3.-Portion of calcium pyrophosphate
crystal (arrow) in a neutrophil. Two clear clefts
(C) containing smaller dense fragments remain
where other crystals had been present. F =
Fibrin-likematerial. x27,OOO.
429
SYNOVITIS OF PSEUDOGOUT
Fig. 4. -Residual lucent
clefts from crystals in phagosome (arrow) of phagocytic
synovial cell. x 15,000.
bers of rod-like Weibel-Palade bodies were
noted in endothelial cells. Crystals were
only occasionally noted in the vicinity of
vessels and in no instance was a crystal
seen in the vessel wall. No crystals were
found in circulating leucocytes.
The alcohol-fixed specimen, as expected,
produced poor tissue fixation for electron
microscopy; but somewhat greater numbers of crystals were preserved and appeared identical to those described above.
Although synovial findings were similar to
those in the Karnovsky fixed material, the
poor fixation yielded no pictures suitable
for publication. Similar crystals were also
seen in the dried frosty-appearing material
obtained at surgery from the third patient
(Fig. 9).
Synovial exudate cells were predominantly polymorphonuclear leucocytes. They
contained frequent crystals identical to
those described above. Many crystals were
clearly in membrane-bounded structures
which also generally contained electrondense material compatible with the appearance of fibrin.
DISCUSSION
The presence of calcium pyrophosphate
crystals identifiable by polarized light in
the superficial synovium, associated with
polymorphonuclear neutrophil infiltration
and edema during acute episodes of pseudogout, has been described by McCarty
et al.lV2 Similar light microscopic findings
were seen in the present case. In addition,
lining cell proliferation, mononuclear infiltrate, and fibrosis, which may be results
of the more chronic aspects of the disease,
were also present.
430
H.RALPH SCHUMACHER, JR.
Fig. 5.-Clear cleft (C) remaining from site of crystal deposit in type B synovial
lining cell.. G =Golgi apparatus, M = Mitochondria. ER = Endaplasmic reticulum.
N = Nucleus. J = Joint space. ~ 2 7 , 0 0 0 .
Fig. 6.-Necrotic neutrophil (arrows) lying in synovial macrophage. C = Lucent
spaces from which crystals were probably dissolved. N = Nucleus of neutrophil.
CF = Collagen fibers. ~25,000.
431
SYNOVITIS OF PSEUDOGOUT
Fig. 7.-Portion of erythrocyte (RBC) enclosed between processes of endothelial cells (E) and pericytes (P) in wall of synovial small blood vessel. J = Closed
junction between endothelial cells. L = Lumen. ~21,000.
The electron microscopic examination allowed some amplification of the histological
findings. The widely varying shapes (most
commonly rhombic or rodlike) and lacelike apparent internal structure of the crystals were identical whether specimens were
fixed in Karnovsky’s fixative, fixed in 95 per
cent alcohol, or merely dried and embedded. This appearance is very similar to the
single electron micrograph of a calcium pyrophosphate crystal published by McCarty
et al. in 19621° and is identical to crystals
seen in the synovium in one case of hemochromatosis with chondrocalcinoskll The
specificity of this crystalline ultrastructure,
however, is not known as we have not yet
had the opportunity to examine other crystals in the electron microscope. The significance of the rare completely electrondense crystal is also unknown. Electron
diffraction was not done.
In most instances not the crystals but
only crystal-shaped clear clefts were seen.
These spaces contained the above crystals
or remnants with sufficient frequency
to establish that these were not different
structures but the sites from which crystals
had been lost in preparation. It appeared
H.RALE'H SCHUMACHER, JR.
432
Fig. 8.-Small
vessel containing erythrocytes (RBC) and neutruphils (PMN) in
the lumen. Process of one neutrophil occupies gap (arrow) between endothelial cells
(E). The endothelium below the gap is fenestrated. Insert is an enlargment of the
region of the gav. LC = Synovial lining cell containing apparent lipid deposits.
J = Joint space. Note absence of crystals demonstrating the focal nature of the crystal
deposition. x4,OOO.
most likely that crystals were lost during
fixation in the water-based fixatives, as they
were no longer identifiable on examination
of thick Epon sections even before exposure
to the electron beam.
As noted above, it is not a new observation that calcium pyrophosphate crystals
can be demonstrated in the synovium.l-6
12,13 Radiologic evidence of cartilage cal-
cification is almost invariably12 most promiment but it has not been established that
initial deposition must be only in cartilage.
Gatter and McCarty" in studying pathological tissue calcifications found calcium
pyrophosphate crystals only in and about
articular structures but did not definitely
incriminate cartilage as the decisive factor.
The distribution of the crystals in the dif-
433
SYNOVITIS OF PSEUDOGOUT
Fig. 9.-Crystals from synovial exudate. The lighter gray material is a supporting
film of Paralodion and carbon. Left x 14,000; Right x70,OOO.
ferent types of synovial lining cells is therefore of interest.
Synovial lining cells have been classified
by Barland and co-workers15 into type A
cells, which are rich in lysosomes and appear to be phagocytic, and type B, presumably synthetic cells, characterized by abundant endoplasmic reticulum. Crystals were
identified in both cell types in this study
as well as in intermediate-type cells with
characteristics of both type A and B cells.
Many synovial crystals were found in obvious phagosomes, suggesting that they
could well have been primarily deposited
in the cartilage and phagocytized by synovial cells only after their release from cartilage. However, the presence of other crystals in apparently synthesizingsynovial cells
also raises the possibility that such cells
are a site of primary intracellular crystal
precipitation. De Shze et aLs have observed
by light microscopy that calcium appears in
chondrocytes before producing intercellular
masses; and McCarty and Gatterl have described the smallest and presumably the
earliest cartilaginous calcium pyrophosphate crystal deposits at the lacunar margins of the chondrocytes. Primary intracellular precipitation of hydroxyapatite has
been described in renal tubular epithelium,* ti
Riddle, Bluhm and Barnhart" in ultrastructural studies of synovial fluid leucocytes have noted the frequent absence of
membranes surrounding urate crystals and
interpreted this as suggesting formation of
the crystals at an intracellular site. In contrast they noted that calcium pyrophosphate
crystals in exudate leucocytes in pseudogout were almost all in large typical membrane-bounded phagosomes.l* Some of the
crystals in this study, both in synovial membrane and exudate cells, had no or incompletely definable surrounding membranes.
The significance of this remains open to
speculation. Membranes could have been
destroyed in uiuo by expanding crystalline
or other components of the phagosome or
during fixation by dissolution of crystals
producing a hyperosmolal solution in the
phagosomes leading to eventual rupture.
The large amounts of cellular debris, including fibrin and loose dense bodies (lysosomes), found intercellularly and in phagosomes suggests that these might contribute,
as has been ~uggested,le-~~
by providing
chemical mediators capable of producing
the increased vascular permeability and
emigration of leucocytes characteristic of
the acute episode of pseudogout. The electron microscopic suggestion of vascular alteration is all compatible with a histaminelike mediation of increased permeability2*
but further study will be needed to assess
the significace of such changes on needle
biopsies. Vascular plugging with platelets
and polymorphonuclear leucocytes and apparent emigration of a leucocyte through
the endothelial wall have been described
in electron microscopic studies of needle
434
H. RALPH SCHUMACHER, J R .
biopsies in Reiter's syndrome by Norton,
Lewis and ZifF."3 No evidence of direct
vascular injury or of close relation of crystals to vessels was seen. Mast cells did not
appear to differ from their normal appearance; but they are known to be capable of
releasing histamine, serotonin and other
potential
mediators
without
visible
changes25
Some of the characteristics noted in this
study had been seen previously in other
diseases and had been considered to be
possibly specific. Phagocytosis of polymorphonuclear leucocytes by synovial macrophages was widespread, suggesting that
this occurs whenever large numbers of cells
are emigrating through proliferated synovium and not only in Reiter's syndrome.23
Similar evidence of phagocytosis has also
been described in the synovial fluid in
pseudogout and gout by Riddle et al.17J8
Focal lipid deposits were often present in
apparent fibroblasts and type B synovial
lining cells, as has been emphasized in
rheumatoid a r t h r i t i ~ .Vascular
~ ~ ~ ~ ~ endo-
thelial rod-like Weibel-Palade bodies
thought by other^^^,^^ to be of possible etiologic significance in rheumatoid arthritis
were seen in many vessels in this study as
well as in normal monkey syn0vium.2~
These studies, therefore, have revealed
characteristic crystals in synovial membrane, suggesting that light and/or electron microscopy of synovial membrane occasionally can be helpful in diagnosis of
pseudogout when no synovial %uid can be
obtained. In addition, many electron microscopic, as well as light microscopic findings, are clearly common to other pathologic processes in the synovium. The findings
have been considered for their possible contribution to further understanding of the
pathogenesis of pseudogout.
ACKNOWLEDGMENTS
The author wishes to acknowledge the frequent
advice of Drs. Guido Majno and Ramzi Cotran of
the Department of Pathology, Harvard Medical
School, and the assistance of Mr. Eduardo Garriga
and Miss Virginia Gilmore in the preparation of the
final prints of the illustrations. Dr. J. Peter Kulka
provided the light microscopy.
SUMMARY
Ultrastructural study of the synovial membrane in a patient with pseudogout showed
calcium pyrophosphate crystals (or the residual clefts from which crystals had been
dissolved) in the interstitium, type A, B and intermediate lining cells, polymorphonuclear leucocytes and deep macrophages.
Cellular debris was found in the interstitium and in phagosomes. Further findings
previously noted in other diseases included vascular endothelial Weibel-Palade bodies,
focal lipid deposits in cells rich in endoplasmic reticulum and phagocytosis of polymorphonuclear leucocytes by macrophages.
SUMMARIO
IN INTERLINGUA
Un studio ultrastructural del membrana synovial in un patiente con pseudogutta
revelava le presentia de crystallos de pyrophosphato de calcium (0 le fissuras ab le
quales ille crystallos habeva essite dissolvite) in le interstitio, in le cellulas de revestimento typo A, B, e intermedie, in leucocytos polymorphonucleari, e in profunde macrophagos.
Detrito cellular esseva trovate in le interstitio e in phagosomas. Constatationes additional (previemente notate in altere morbos) includeva corpores de Weibel-Palade
vasculo-endothelial, focal depositos lipidic in cellulas ric in reticulo endoplasmatic, e
phagocytose de leucocytospolymorphonucleariper macrophagos.
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SYNOVITIS OF PSEUDOGOUT
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