Vol. 47, No. 2
Printed in U.S.A.
T H E AMERICAN JOURNAL OP CLINICAL PATHOLOGY
Copyright © 1067 by The Williams & Wilkins Co.
CYTOLOGY OF SYNOVIAL FLUID IN RHEUMATOID ARTHRITIS
THEODORE I. MALININ, M.D., THOMAS J. PEKIN, JR., M.D., AND
NATHAN J. ZVAIFLER, M.D.
Departments of Research Pathology and Medicine, Georgetown University Schools of Medicine
and Dentistry, Washington, D. C. 80007
The cytologic examination of synovial
fluid in rheumatoid arthritis (RA) is not a
routine procedure in most clinical laboratories, despite the fact that inflammatory
articular effusion is a frequent occurrence in
this disease. Laboratory examination of the
synovial fluid in RA is usually limited to
white blood cell count, characterization of
the mucin clot, and possibly tests for rheumatoid factor and hemolytic complement
activity. The demonstration of specific crystals in wet preparations of synovial fluid in
gouty arthritis and chondrocalcinosis is of
definite diagnostic value, as is bacteriologic
examination in cases of infectious arthritides.
Reports that vacuoles observed in polymorphonuclear leukocytes in wet preparations of
synovial fluid are peculiar to rheumatoid
arthritis have not been confirmed.1"3 The
present study was undertaken in the hope
that systematic cytologic examination of
synovial effusions from patients with rheumatoid arthritis would disclose some consistent findings unique to this disorder.
The presence of peculiar Feulgen-positive
particles in rheumatoid synovial fluid has
been reported in a preliminary communication.4
MATERIALS AND METHODS
Synovial fluid was obtained by needle
aspiration of the suprapatellar pouch from 25
patients with clinically established classic or
definite rheumatoid arthritis. When present,
the fluid was also obtained from elbows.
Seven patients in this group were subjected
to repeated aspirations. A total of 39 individual rheumatoid fluid specimens were exReceived, March 18, 1966.
This study was aided by grants from the
National Foundation, United States Public
Health Service (AM 05042 and 05140), and the
Arthritis Foundation.
amined. The cell counts of the fluids varied
between 10,000 and 50,000 nucleated cells
per cu. mm.
For comparison, synovial fluid was obtained from IS patients with knee effusions
resulting from causes other than rheumatoid
arthritis. The diagnoses in this group of
patients were as follows: Reiter's syndrome
(five patients), gouty arthritis (three patients), infectious arthritis (one patient with
gonococcal arthritis, one patient with probable gonococcal arthritis, and two patients
with tuberculous arthritis), chondrocalcinosis (two patients), juvenile rheumatoid arthritis (three patients), and osteoarthritis
(one patient). Except in osteoarthritis, synovial white blood cell coimts in the cases
were comparable to those of rheumatoid
arthritis.
Slide films of synovial fluids were prepared
immediately after aspiration, and were airdried and stained unfixed. The smears were
stained with Wright-Giemsa, Maximow's
hematoxylin eosin azure II, periodic acidSchiff (PAS) reaction, fat stains, methyl
green-pyronin for ribonucleic acid, Feulgenoxidized tannin-azo technic (FOTA) for concomitant demonstration of deoxyribonucleic
acid (DNA) and proteins, and toluidine blue
for metachromasia. Staining for nucleic acids
was controlled by incubation with deoxyribonuclease, and ribonuclease, and omission
of hydrolysis with hydrochloric acid. Staining for protein was controlled by omission of
oxidation with periodic acid.
RESULTS
The predominant cells in all preparations
of synovial fluid from patients with rheumatoid arthritis were mature polymorphonuclear granulocytes with the usual appearance. These accounted for from 75 to 90 % of
all nucleated cells. The remainder of the cell
population was comprised of lymphocytes (5
to 10%), monocytes, occasional macrophages
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204
MALTNIN ET
with eccentric round nuclei, and large degenerating cells with poorly defined nuclei.
In about 1 % of rheumatoid synovial
granulocytes there was a displacement of the
nuclear chromatin to the periphery of the cell
(Fig. 1). The center of such cells was homogeneous azure-gray in Maximow-stained
preparations, and pale blue in WrightGiemsa preparations. The material in the
center, however, was often clearly outlined
in FOTA preparations where it stained
yellow-brown, indicating its protein nature
(Figs. 2 and 3). No specific coloration was
observed with other stains. The cells with
nuclear displacement and protein masses in
the center were encountered in 22 of 25 cases
of rheumatoid arthritis.
Polymorphonuclear granulocytes with
multiple segregated nuclear chromatin accounted for about 2 to 5 % of all granulocytes
in rheumatoid synovial fluid. These cells
resembled the so-called hypersegmented polymorphonuclear granulocytes usually observed in inflammatory exudates, and in the
peripheral blood of patients with pernicious
anemia. The nuclei of these granulocytes
frequently did not stain uniformly with
Schiff's reagent. In such instances the staining was more intense at the periphery and
often only faint in the center of the nuclear
chromatin (Fig. 4).
Small cells with one or more nuclear-like
structures and frequently Feulgen-positive
intracytoplasmic globules were present in
rheumatoid synovial fluid. The number of
these cells varied from case to case, but they
usually did not exceed 1 % of total nucleated
cell numbers. The nuclear structures in
these small cells were uniformly dark
red in FOTA preparations. The cytoplasm
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was fuchsinophilic in smears stained with
Masson trichrome stain. Figure 5 shows such
a cell, as well as a polymorphonuclear granulocyte with segregated nuclei.
Round and oval, basophilic, Feulgenpositive, globular particles were consistently
found in the cytoplasm of rheumatoid polymorphonuclear granulocytes, occasionally in
mononuclear cells, and in the small cells
mentioned above. Examples of these globules
are shown in Figures 5 to 8. In rare instances
the cell contained numerous globules and
only one or two structures suggestive of the
nucleus (Fig. 7). The staining intensity of the
globules varied only a little, and they frequently stained much darker than the
nuclei of cells in which they were found.
Numerous extracellular DNA globules
were found throughout the rheumatoid synovial fluid in all cases of rheumatoid arthritis
(Fig. 9). In the majority of instances, the
free-floating DNA globules were comprised
exclusively of Feulgen-positive material, but
in some cases the globules were surrounded
by a rim of tannophilic protein that gave
them a "miniature lymphocyte" appearance.
The extracellular globules were found in
several locations: away from the cells as
illustrated in Figure 9, in close proximity to
cells, immediately adjacent to the cell membrane (Fig. 10), and in the pseudopod-like
projection of polymorphonuclear granulocytes (Fig. 11).
Figure 11 also illustrates the difference in
the staining intensity between the nucleus of
the cell and the intracellular globule. This
was observed in many instances. Because of
such discrepancy in staining intensity it
seemed to be unlikely that these globules
were being extruded from the cells. It would
FIG. 1 (upper). Polymorphonuclear granulocytes from rheumatoid synovial fluid. Note a granulocyte with peripheral displacement of nuclear chromatin (arrow). Maximow's hematoxylin, eosinazure II. X 400.
FIG. 2 (middle, left). Polymorphonuclear granulocyte with marked peripheral displacement of
the nucleus. The cytoplasm of this cell contains a round mass of weakly tannophilic protein. The
nucleus at the periphery stained red and the protein mass dark yellow-brown. Feulgen-oxidized
tannin-azo (FOTA). X 1100.
FIG. 3 (center). Polymorphonuclear granulocyte with a strongly tannophilic (dark brown) mass
in the center. FOTA. X 1500.
FIG. 4 (middle, right). Polymorphonuclear granulocyte with unevenly staining nuclear chromatin. FOTA. X 1000.
FIG. 5 (lower, left). A small peculiar cell adjacent to a polymorphonuclear granulocyte with
multilobular nucleus. The nuclear structures in the small cell stained dark red. Note that it also
contains two small dark red globules in the cytoplasm (arrow). The cytoplasm itself stained dark
brown, indicating high protein content. FOTA. X 1000.
FIG. 6 (lower, right). A polymorphonuclear granulocyte with intracytoplasmic Feulgen-positive
globules. FOTA. X 1400.
Feb. 1967
CYTOLOGY OF SYNOVIAL FLUID IN RA
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206
MALININ ET
FIG. 7. Cell with three large nuclear structures
and three small DNA globules. FOTA. X 1000.
seem more probable that they were being
phagocytized by the polymorphonuclear
granulocytes.
In rare instances adherence of amorphous
Feulgen-positive material to the cell membrane of the polymorphonuclear neutrophilic
leukocytes was observed. In two cases of
rheumatoid arthritis, normal-appearing polymorphonuclear granulocytes were found to
be phagocytized by mononuclear cells, as
demonstrated in Figure 12.
Examination of synovial fluid from IS
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AL.
nonrheumatoid cases revealed no changes
similar to those consistently found in rheumatoid fluids in 15 of the specimens. Fluid
from one case of acute gouty arthritis contained extracellular, Feulgen-positive globules and small intracellular particles, as did
one fluid specimen from a patient with
Reiter's disease. Elongated particles of Feulgen-positive material and large extracellular
DNA globules were present in one case of
probable gonococcal arthritis with a white
cell count of 60,000 mm. Phagocytosis of
polymorphonuclear granulocytes by macrophages was quite prominent in acute stages
of Reiter's disease.5
Peripheral nuclear displacement and the
adherence of Feulgen-positive material to
the cell membrane were not present in nonrheumatoid synovial fluids.
DISCUSSION
Peculiar cytologic changes were consistently observed in the rheumatoid synovial
fluid leukocytes. These changes consisted of
nuclear fragmentation, presence of intracellular and extracellular DNA particles,
\
FIG. 8 (left). A small cell with dark nucleus and round intracytoplasmic globule. FOTA. X 900.
FIG. 9 (right). Two extracellular Feulgen-positive globules. FOTA. X 900
Feb. 1967
CYTOLOGY OF SYNOVIAL FLUID IN RA
*
F I G . 10. D a r k l y staining Feulgen-positive globules adjacent to the cell membrane of a polymorphonuclear granulocyte. F O T A . X 1100.
adherence of amorphous DNA material to
the cell membrane, and peripheral displacement of nuclear chromatin with accumulation of protein in the center of the cell. The
presence of an occasional extracellular and
intracellular DNA particle was observed in
three of IS nonrheumatoid cases. Phagocytosis of polymorphonuclear granulocytes
by mononuclear cells was also noted; this is
apparently a phenomenon generally encountered in inflammatory exudates. 6,7
207
The origin of the extracellular DNA particles is obscure; however, the finding of the
DNA particles surrounded by protein, at
times resembling "miniature lymphocytes,"
tends to implicate DNA-protein complexes.
The striking difference in the Feulgen staining intensity between the nuclei and the
DNA globules found in the cytoplasm and
in the pseudopod-like projections of polymorphonuclear granulocytes suggests that
DNA material is being phagocytized, but
not extruded, by these cells.
Polymorphonuclear granulocytes with
multiple, segregated, unevenly staining, nuclear lobules probably represent old cells in
the early stages of autolysis. It seems unlikely that after disruption of these cells the
pale nuclear chromatin can undergo condensation sufficient to produce brightly colored,
uniformly staining DNA particles. Retention
of protein around the nuclear remnants of a
dead cell also would be difficult to explain.
On the other hand, polymorphonuclear
granulocytes with multiple dark staining
nuclear bodies, if fragmented, may well
produce peculiar cell-like structures and extracellular DNA particles. These cells are
also probably in the end stage of their life
span, but their nuclear DNA is condensed.
•
F I G . 11 (left). A darkly staining Feulgen-positive globule in the pseudopod-like projection of
a polymorphonuclear granulocyte. Note the difference in the staining intensity of the globule and
the nucleus of the cell. F O T A . X 1000.
F I G . 12 (right). An intact polymorphonuclear leukocyte phagocytized by a mononuclear cell.
Maximow's hematoxylin, eosin azure I I . X 1000.
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MALININ ET
The presence of peculiar small cell-like
structures with intensely colored, nuclearlike bodies, DNA particles, and fuchsinophilic cytoplasm suggests a possibility that
extracellular DNA particles may also originate in these cells. A sudden breakdown of
these cells could result in extracellular DNA
globules surrounded by protein. Histogenesis
of these cells has not been investigated, but
their peculiar appearance certainly sets them
apart from usual leukocytes.
Although DNA particles were observed in
all synovial fluids from 25 patients with
rheumatoid arthritis, this finding does not
seem to be specific for rheumatoid arthritis,
as these particles were also found in three
of IS nonrheumatoid conditions. Quantitatively, the particles were more numerous and
consistently present in rheumatoid synovial
fluids; it is thus unlikely that DNA particles
are merely byproducts of inflammation of a
joint.
Displacement of the nuclear chromatin to
the periphery of the cell occurred when the
center of the cell was occupied by either a
large DNA globule or a protein mass (Figs. 2
and 3). Nuclear displacement by a protein
mass was encountered only in cases of rheumatoid arthritis. This may then be the
so-called "rheumatoid cell" recently demonstrated by Williamson and Ling8 in in vitro
experiments. The observations of Hollander
and co-workers2 on the presence of 7S and
19S 7-globulins in leukocytes from rheumatoid synovial fluids may also relate to these
same cells.
The origins of the observed cytologic
abnormalities in rheumatoid synovial effusions are still obscure and their exact significance awaits definition, but cytologic examination of synovial fluid as an aid in the
diagnosis of rheumatoid arthritis appears to
be promising. In no other disease of the joints
studied to date has the combination of
peripheral displacement of nuclear chroma-
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AL.
tin by proteinaceous material and intra- and
extracellular Feulgen-positive bodies been
seen.
SUMMARY
Specimens of synovial fluid from 25 patients with rheumatoid arthritis and 18
patients with nonrheumatoid diseases were
examined cytologically. Peculiar changes
were noticed in cells in fluid from rheumatoid
arthritis. These changes consisted of nuclear
fragmentation, intra- and extracellular deoxyribonucleic acid (DNA) bodies, adherence
of amorphous DNA material to the cell, and
peripheral displacement of nuclear chromatin with accumulation of protein in the
center of the cell.
The presence of an occasional extracellular
and intracellular DNA particle was observed
in three of 18 nonrheumatoid cases.
REFERENCES
1. Astorga, G., and Bollet, A. J.: Diagnostic
specificity and possible pathogenic significance of inclusions in synovial leukocytes.
Arthritis Rheum., 8: 511-523, 1965.
2. Hollander, J. L., McCarty, D. J., Astorga, G.,
and Castro-Murillo, E.: Studies on the
pathogenesis of rheumatoid joint inflammation. I. The "R. A. Cell" and a working
hypothesis. Ann. Int. Med., 62: 271-280,
1965.
3. Malinin, T. I., Pekin, T. J., Bauer, H., and
Zvaifler, N. J.: Vacuoles in synovial fluid
leukocytes. Am. J. Clin. Path., 46: 728-731,
1966.
4. Malinin, T. I., Pekin, T. J., Zvaifler, N. J., and
Bauer, H.: Intracytoplasmic and extracellular particles in rheumatoid synovial fluid.
Arthritis Rheum., 7: 743, 1964 (Abstr.).
5. Pekin, T. J., Malinin, T. I., and Zvaifler, N. J.:
Studies on synovial fluids in Reiter's disease.
Clin. Res. 14: 56, 1965 (Abstr.)
6. Riis, P.: The Cytology of Inflammatory Exudate. Munksgaard, Copenhagen, 1959.
7. Speirs, R. S.: How cells attack antigens.
Scientific Am., 210: 58-64, 1964.
8. Williamson, N., and Ling, N. R.: Cellular
reaction to complexes formed between rheumatoid factor and aggregated human gamma
globulin. Ann. Rheum. Dis., 24: 513-521,
1965.