Absence of histologic evidence of synovitis in patients with Gulf War veterans' illness with joint pain.

OBJECTIVE: An unexplained multisymptom illness, Gulf War veterans' illness (GWVI), has been described among allied force veterans of the first Gulf War (1990-1991). It has been proposed that some of its symptoms reflect an immune dysfunction, and rheumatologic symptoms including joint pain and stiffness are reported frequently. However, it is unknown whether synovial inflammation causes the articular symptoms. We examined synovial tissue from individuals with GWVI and joint pain for evidence of inflammation. METHODS: We compared synovial biopsy samples from 6 individuals with GWVI and joint pain with samples from 9 clinically asymptomatic controls (hematoxylin and eosin [H&E] stains only) and biopsy samples or surgically obtained specimens from 10 patients with rheumatoid arthritis (RA) and 12 with osteoarthritis (OA). Inflammatory changes were quantified in H&E stained sections with a modified synovitis score by immunostaining for CD3, CD20, CD38, CD68, Ki-67, and von Willebrand factor, and with a composite inflammation score based on these markers. RESULTS: Normal histology was seen in the GWVI specimens, except for mild focal lining hyperplasia and rare low-grade perivascular infiltrates in 1 specimen each. Mean +/- SEM synovitis scores were lowest and nearly identical in control (1.38 +/- 0.30) and GWVI specimens (1.41 +/- 0.29), intermediate in OA specimens (2.64 +/- 0.39), and highest in RA specimens (6.0 +/- 0.19). Likewise, inflammatory cells, cell division, vascular density, and composite inflammation score were lowest in the GWVI specimens. CONCLUSION: Despite significant joint pain, the GWVI synovia did not differ from normal controls. These results agree with other studies that have failed to document inflammatory or immunologic etiologies in GWVI.

Detection and initial characterization of synovial lining fragments in synovial fluid.

OBJECTIVE: Free fragments of synovium have occasionally been seen in synovial fluid but have not been studied systematically. We wished to establish a method for the reliable detection of these fragments in joint and bursa effusions and begin to characterize them by histochemical and immunohistochemical methods. METHODS: Cell smears, wet drop preparations and cytospins were prepared from 39 consecutive joint and bursa effusions. Paraffin cell blocks were prepared from a subset. Analysis encompassed standard and polarized light microscopy, histochemistry, immunohistochemistry and transmission electron microscopy (EM). Synovial biopsy tissue from one different patient was examined for comparison. RESULTS: Tissue fragments were not seen in Wright-stained cell smears and only rarely in wet drop preparations. In contrast, variously sized fragments with the histological appearance of hyperplastic synovial lining were detected in ethanol-fixed, haematoxylin/eosin-stained cytospins from bursitis and all arthropathies studied [17/24 (71%) of non-inflammatory and 12/15 (80%) of inflammatory specimens]. Immunostaining revealed CD68 expression in a subset of cells in a pattern characteristic of hyperplastic synovial lining. Juxtaposed cells with morphological features of macrophage-like and fibroblast-like synoviocytes were seen by EM. CONCLUSIONS: Synovial lining fragments can be detected in effusions from diverse arthropathies and bursitis. They maintain important properties of the synovial lining and can be analysed by immunohistochemistry. They may afford the opportunity to study a relatively pure preparation of synovial lining cells without the need for cell culture, and to evaluate their possible role in augmenting or perpetuating synovitis or joint damage.

Intra-articular corticosteroid preparations: different characteristics and their effect during inflammation induced by monosodium urate crystals in the rat subcutaneous air pouch.

OBJECTIVE: To examine the effects of three commonly used intra-articular depot corticosteroid preparations tested in a rat air pouch model and their effect against monosodium urate (MSU) crystal-induced inflammation. Rheumatologists use intra-articular corticosteroid preparations to relieve pain and inflammation of acute monoarthritis without really knowing their effects on the synovial fluid and membrane or the differences between distinct preparations. This work compares the effect of three commonly used corticosteroid preparations in vivo, showing that they behave differently. METHODS: A subcutaneous air pouch was formed in male Sprague-Dawley rats. A first group of 6-day-old air pouches were injected with 10 ml of 6 mg/ml normal saline solution, 6 mg/ml betamethasone containing both depot betamethasone acetate and soluble betamethasone phosphate (Celestone) in 9 ml of normal saline solution, 20 mg/ml of prednisolone tebutate (Hydeltra) in 9 ml of normal saline solution or 20 mg/ml of triamcinolone hexacetonide (Aristospan) in 9 ml of normal saline solution. A second group (group 2) of air pouches were injected with 15 mg of synthetic MSU crystals and 24 h later they were reinjected with 1 ml of the same three corticosteroid suspensions. For each condition four rats were killed at 6, 24, 48 h and 7 days. Pouch fluid and tissue were analysed. RESULTS: In the first 6 h after normal saline solution or corticosteroid injection into the air pouch there were mildly increased leucocyte counts in the air pouch fluid. Betamethasone-injected pouches showed no cells in the fluid after 6 h and no crystals after 24 h, triamcinolone-injected pouches still showed rare cells at 7 days. Both triamcinolone and prednisolone crystals persisted in higher numbers and lasted longer in the fluid than did betamethasone (P<0.05). In group 2 MSU crystal phagocytosis in the fluid was decreased in the betamethasone- (P<0.01), prednisolone- (P<0.003) and triamcinolone- (P<0.006) injected pouches when compared with the MSU crystal-injected pouches alone. Pouches injected with MSU crystals alone showed the most intense tissue inflammation at all times. After MSU, betamethasone-injected pouches had a rapid but mild decrease in the number of lining cells and inflammation. In contrast, triamcinolone- and prednisolone-injected pouches showed a very thin tissue with few or no vessels and almost no inflammation at 7 days. The pouches injected with MSU crystals and any of the corticoid preparations had three times more tophus-like structures and persistent crystals identified than the ones injected with MSU crystals alone. CONCLUSION: Each of the corticosteroid preparations by themselves produced very mild transient inflammation. The betamethasone preparation with a soluble steroid component had a quicker but milder anti-inflammatory effect on MSU crystal-induced inflammation. In contrast to the doses used, prednisolone tebutate and triamcinolone hexacetonide preparations dramatically suppressed urate crystal-induced inflammation at 7 days, but both produced atrophy and necrosis of the membrane, yielding a very thin membrane with almost no vessels. When used for MSU crystal-induced inflammation these corticosteroid preparations suppressed some aspects of inflammation but may actually promote the persistence of MSU crystals and the formation of tophi.

Synovial fluid features and their relations to osteoarthritis severity: new findings from sequential studies.

OBJECTIVE: Many factors are involved in the osteoarthritic process. It is not yet known which are initiators, promoters or simply results. Thus, we have evaluated some of those potentially important factors in osteoarthritis (OA) as observed sequentially for the first time in synovial fluids. DESIGN: Synovial fluids (SF) obtained between 1992-2002 were all routinely evaluated for gross appearance, leukocyte counts and microscopic examination of wet drop preparations. We used regular and polarized light and alizarin red s stains. We separated out all OA patients, then we looked for patients who had more than two synovial fluid analyses to get sequential information. Time between first and final aspiration ranged from 2 to 7 (3.6+/-1.6) years and number of analyses per patients from 3 to 6 (3.3+/-0.7). We related synovial fluid crystals, fibrils and white blood cell count (WBC) to age, sex, disease duration and radiographic assessment according to the Kellgren-Lawrence radiographic rating system. RESULTS: Of 4523 synovial fluid examinations, we found 855 in patients with knee OA; 330 patients with adequate clinical details for comparison were included in our study. Twenty-six patients (one woman and 25 men) had sequentially examined SF. We found that 52% of those OA patients with effusions studied had crystals identified in their synovial fluid. Twenty-one percent of all the patients had CPPD crystals, 47% had hydroxyapatite, also called basic calcium phosphate (BCP) crystals and 16% had both types of crystals. Microscopically identifiable fibrils were found in 60% of SF. In sequentially examined patients, CPPD crystals and apatite (BCP) were found in 19% and 23%, respectively, at the first aspiration and, in 34% and 58% at the final aspiration. Fibrils were seen in 54% at first examination and 85% later. Apatite and fibrils showed more significant correlation with time (r=0.51,r =0.92) than did CPPD (r=0.32). SF WBC correlated only with CPPD crystals and did not increase with OA duration or severity. CPPD, apatite and fibrils all correlated with higher radiographic grades of OA. CONCLUSIONS: As noted before CPPD and apatite crystals were more common in patients with more severe OA. New findings are that our sequential cases showed that there were some patients with no crystals at onset but that crystals appeared with progression of the disease. Fibril presence in SF also correlated with progression of the disease.

Calcium apatite crystals in synovial fluid rice bodies.

BACKGROUND: Rice bodies can occur in the joints in many rheumatic conditions, but they are most common in rheumatoid arthritis. They are generally believed to occur rarely in patients with osteoarthritis, but one study reported rice bodies with apatite crystals. OBJECTIVE: To report on a series of joint fluids with rice bodies containing apatite clumps and examine their clinical pictures. METHODS: All synovial fluid analysis reports for 10 years were reviewed for rice bodies and eight patients were reported on. A series of patients with a variety of diseases with synovial fluid rice bodies found to contain calcific material is described. All were examined by compensated polarised light and alizarin red stain, and four were examined by electron microscopy. RESULTS: The eight patients all had alizarin red S chunks embedded throughout the rice body. Transmission electron microscopy disclosed the presence of a matrix of collagen, fibrin, and amorphous materials containing typical apatite crystals. Clinical diagnoses, radiographic findings, and leucocyte counts varied, but six of the eight patients had had previous repeated corticosteroid injections into the joints. CONCLUSION: Aggregates of apatites may be more common than previously recognised in rice bodies as they are not routinely sought. Whether they are a result of joint damage or depot steroid injections and whether that might contribute to further joint injury now needs to be investigated.

Treatment of chronic gout. Can we determine when urate stores are depleted enough to prevent attacks of gout?

OBJECTIVE: To determine if lowering of serum uric acid (SUA) concentrations below 6 mg/dl or longer duration of lowered SUA will result in depletion of urate crystals from the knee joints and prevent further attacks of gout. METHODS: A prospective study was initiated 10 years ago at Philadelphia VA Medical Center to attempt to maintain SUA levels of patients with crystal proven gout at < 6.0 mg/dl. We recalled all 57 patients who were available during 1999. Patients were divided into 2 groups: Group A, with SUA still > 6 mg/dl, and Group B, with SUA < or = 6 mg/dl. A knee joint aspirate was requested from all asymptomatic Group B patients and many in Group A. Aspirates were examined by polarized light microscopy for identification of crystals. RESULTS: There were no differences between the groups in age, sex, duration of gout, or serum creatinine. Group A (n = 38) had a mean of 6 attacks of gout for the recent year, those with tophi having the most frequent attacks. Among the 16 patients in this group who agreed to knee aspiration, monosodium urate (MSU) crystals were found in 14, although they were asymptomatic at the time. Nineteen patients (Group B) were able to maintain serum urate levels < or = 6 mg/dl for > 12 months. Nearly half of them had no attack of gout for 2 or more years, with a mean of 1 attack in the last year for the whole group. Three patients in whom tophi were found did not have major flares of gout within the past year. Knee joint aspiration was done on 16 asymptomatic patients. Seven (44%) still had MSU crystals present in their knees. Patients in this group who were taking prophylactic colchicine did not differ with respect to the character of synovial fluid from those who had discontinued it for up to several years, although the frequency of attacks was less in those who continued colchicine. CONCLUSION: A majority of patients were able to deplete urate crystal stores in their knee joint fluids when their SUA levels were kept to < or = 6 mg/dl for several years. The mechanisms for persistence in some patients, and whether such crystals have clinical implications, are not known. Patients with chronic gout need serum urate concentrations to be kept low to prevent further attacks.

Processing of noninflammatory synovial fluids with hyaluronidase for cytospin preparations improves the accuracy of differential counts.

Differential leukocyte counts on noninflammatory synovial fluids (NISF) are not widely reported or used in research, apparently due to technical difficulties related to either high viscosity or low numbers of cells. We describe an evaluation of a technique using hyaluronidase and cytospin preparations to study NISF. Twenty-three consecutive synovial fluids (SF) with less than 2,000 white blood cells (WBC)/mm3 were studied either by the usual smear of a single drop or by adding two drops of hyaluronidase (150 USP units/ml) to 0.25 cc of SF and cytocentrifuging at 800 rpm for 10 min. Both preparations were stained with Wright's stain. Cytospin preparations gave better morphology, and in 22/23 specimens we could count 100 cells on one slide. Smeared preparations gave dark cells and required 2-3 slides to count 100 cells. Differential counts on the cytospin preparations consistently showed higher percentages of monocytes, suggesting that these cells were underdetected and misinterpreted as lymphocytes on the routine smears. Polymorphonuclear leukocytes (PMN) were significantly less frequent (P 0.005) in osteoarthritis (OA) fluids than in the other diseases with NISF. Relatively more PMN may suggest consideration of a diagnosis other than OA. Cytospin preparations of hyaluronidase-treated NISF may open up an important area for investigation of the role of SF cells in less inflammatory diseases.

Morphometric studies on synovium in advanced osteoarthritis: is there an association between apatite-like material and collagen deposits?

OBJECTIVE: To look for the frequency and the influence of apatite-like deposits in the synovial membrane of advanced osteoarthritic joints. METHODS: Synovium of 16 joints undergoing total arthroplasty for advanced primary osteoarthritis was embedded in paraffin. Adjacent sections were stained with alizarin red S, Mowat's pentachrome, Hematoxylin-eosin and Gomori to show apatite-like deposits, collagen, cartilage fragments, vessels, cells and iron respectively. Histomorphometry was carried out for the apatite-like and collagen deposits by the point counting method; the density of vessels and cells was also quantified. RESULTS: 14 out of the 16 specimens contained apatite-like material, mostly on the synovial surface or just beneath. There was no correlation between the apatite-like deposits and any other measured histological parameter (fibrosis, villus length, synovial lining cell width, density of synovial vessels or cells). Interestingly, the p value for a correlation between the amounts of apatite and collagen deposits was close to significance (r = 0.53; p = 0.055) when the relative volume of the apatite-like material was less than 1.2% (n = 13). CONCLUSIONS: Apatite-like deposits are frequently observed in the synovium of late stage osteoarthritis. Although not statistically significant, these results suggest a possible association between apatite-like and collagen deposits when the amount of apatite deposits is low. Further quantitative studies are recommended to investigate this observation.

Effect of a nonsteroidal antiinflammatory drug on synovial fluid in osteoarthritis.

OBJECTIVE: The use of nonsteroidal antiinflammatory drug (NSAID) therapy in osteoarthritis (OA) is controversial because of suggestions that pure analgesics can be as effective as NSAID for pain relief. In addition, there is incomplete information whether antiinflammatory effects have any longterm benefit in OA. NSAID have been known to affect synovial fluid (SF) prostaglandins in rheumatoid arthritis. We describe the first examination of the effect of an NSAID, etodolac, on SF prostaglandins, cytokines, and cells in OA. METHODS: Joint fluids were studied before and 2 weeks after initiation of therapy with etodolac 400 mg tid. Leukocyte counts, prostaglandin, interleukin 6, and tumor necrosis factor were measured. RESULTS: Pretreatment features of SF did not predict clinical response. We found no change in the relatively low leukocyte counts. However, SF prostaglandin levels and interleukin 6 levels were significantly decreased and tumor necrosis factor alpha levels were increased after therapy with NSAID. CONCLUSION: This NSAID had potentially important local effects that could be either beneficial or deleterious. Further studies on effects of this and other NSAID on a broader variety of SF and synovial cytokines may help predict longterm effects of NSAID on progression of OA.

Idiopathic destructive arthropathies: clinical, light, and electron microscopic studies.

Rapidly destructive arthritis with relatively noninflammatory joint fluids has been described at shoulders as well as at other large joints under a variety of names in older persons. Because this syndrome frequently is not recognized, we have reviewed our experience with such cases. After excluding patients with known causes such as avascular necrosis, Paget's disease, neuropathic arthropathy, endocrine disorders, radiographic evidence of chondrocalcinosis, and hemodialysis, we have seen 20 such patients who are described in this report. All were over age 67, and 16 were women. Seventeen patients had shoulders involved; 6 had destructive arthritis at the knees. Many other patients had multiple sites affected. Six patients also had erosive osteoarthritis of small joints.All patients had diffuse cartilage loss and juxta-articular bone destruction. Patients had been symptomatic a mean of 3.5 years at the time of the study. Bony sclerosis on x-ray was more common than reported in previous series. Joint effusions were bloody or clear. All fluids had strongly positive alizarin red S positive chunks; apatites were confirmed in all 7 studied by electron microscopy. Nine had calcium pyrophosphate dihydrate crystals. The latter were somewhat more commonly associated with bony sclerosis, but crystal types did not explain all differences seen in x-ray patterns.Destructive arthropathy of the elderly involves many large joints in addition to the shoulders, and some patients also have erosive osteoarthritis of fingers. Although calcium pyrophosphate dihydrate crystals were present in many patients along with apatites, the role of crystals is not clear. Radiographic patterns may be more diverse than previously suggested by some researchers, so that the spectrum of idiopathic destructive arthritis reported may depend on selection criteria. It is important to be aware of this noninfectious cause of severe joint destruction at a variety of joints.

Calcium pyrophosphate dihydrate crystal deposition in synovium. Relationship to collagen fibers and chondrometaplasia.

OBJECTIVE: Reasons for apparent primary deposition of calcium pyrophosphate dihydrate (CPPD) crystals in some synovial membranes have not been systematically examined. We undertook the present study to investigate for and compare possible cellular and matrix factors related to the presence of these crystals in synovium and cartilage. METHODS: Ten synovial membrane specimens and 6 cartilage specimens were obtained at the time of joint surgery from 10 patients with CPPD crystal deposition disease, for light microscopic (LM) and electron microscopic (EM) studies. RESULTS: In all synovial and cartilage specimens, we found many of the small CPPD crystals aligned on or in parallel to collagen fibers, as seen by EM. In 9 of the 10 crystal-containing synovia, we found foci of chondrometaplasia adjacent to CPPD, by LM. In 7 of the synovia, including the one without LM evidence of chondrometaplasia, we observed the presence of chondrocyte-like cells by EM. We did not note any predictable relationship between the crystals and matrix vesicles, either in synovium or in cartilage. CONCLUSION: Our EM findings provide evidence of the relationship of small CPPD-like crystals, presumably early forms, to collagen fibers both in synovium and in cartilage. By LM and EM, we also demonstrate evidence of a close association between chondrometaplasia and CPPD deposits in synovium. We suggest that chondrometaplasia might be responsible for synovial CPPD formation in predisposed patients. Both the collagen fibers and chondrocyte-like cells seem to be involved in the primary formation of CPPD deposits in the synovium as well as in the cartilage.

A comparison of five preparations of synthetic monosodium urate monohydrate crystals.

We conducted preliminary crystallographic investigations and biologic studies on 5 synthetically grown preparations of monosodium urate monohydrate (MSUM) crystals including one preparation of urate spherulites. The 5 crystal preparations exhibited wide variations in morphology, size, surface area, and ultrastructure, but only a few changes in their biologic effects. When injected intraarticularly, urate spherulites were less phlogistic than most acicular forms. Since crystal-cell interactions and inflammatory responses are influenced by crystal size, morphology, and surface characteristics, standardization of methods of preparing MSUM crystals is, therefore, important, particularly when analyzing and comparing results from different research laboratories.

Estimated synovial fluid leukocyte numbers on wet drop preparations as a potential substitute for actual leukocyte counts.

Leukocyte counts on synovial fluid are widely used in classification of joint disease as inflammatory or "noninflammatory." We have evaluated estimates of leukocytes (WBC) per high power field (HPF) on wet drop preparations to see if they might be suitable substitutes in some situations. If there was a mean WBC of 0-2/field on 10 HPF, all leukocyte counts were less than 1,300/mm3. Thus, in such fluids expense might be reduced by eliminating the actual leukocyte count. Estimates also roughly correlated with WBC counts at higher counts but were less consistent.

Supravital staining of cells in noninflammatory synovial fluids: analysis of the effect of crystals on cell populations.

Leukocyte differential counts are rarely performed on synovial fluids (SF) with low leukocyte (WBC) counts as they have been difficult to do with standard Wright stains. We performed SF analysis including supravital (SV) stains for differential counts on 100 consecutive relatively noninflammatory synovial fluids defined as any SF with less than 2,000 cells/mm3. SV stains gave clear cellular morphology. Monocytes were the most prominent cells in all noninflammatory synovial fluids. Thirty-eight fluids were found to have crystals (monosodium urate (MSU) in 15, calcium pyrophosphate dihydrate (CPPD) in 5, CPPD plus apatite-like crystals in 9, apatite-like clumps alone in 8 and lipid liquid in 1). WBC counts, percentages of polymorphonuclear (PMN) and absolute PMN counts were greatest in fluids with MSU or CPPD crystals. Fluids with apatite-like clumps alone tended to have the lowest WBC counts. The presence of WBC over 500 cells/mm3, over 20% PMN and absolute PMN counts over 250 cells/mm3 should encourage a careful search for crystals, especially MSU, in noninflammatory synovial fluids.

Development and evaluation of a method for preservation of synovial fluid wet preparations for quality control testing of crystal identification.

We describe a technique for embedding drops of fresh synovial fluid in mounting resin to prepare test slides that retain crystal morphology for at least 2 weeks. Hospital and private rheumatologists were solicited nationwide to test themselves and their staff with 5 unknown specimens. Eighty-nine percent identified monosodium urate (MSU) crystals from a tophus but only 71% identified smaller and less frequent MSU. Seventy-five percent correctly identified calcium pyrophosphate dehydrate and 42% detected steroid crystals. Apatite crystal clumps included as control slides with no birefringent material were suspected by only 7%. This or another method which simulates actual practice by also testing the equipment and handling of the microscope is proposed as an important component of quality control testing programs for synovianalysis.

Effect of joint motion on experimental calcium pyrophosphate dihydrate crystal induced arthritis.

We studied the effects of joint movement and immobilization on acute and chronic calcium pyrophosphate dihydrate (CPPD) crystal induced arthritis in lapine knee joints. Exercised CPPD injected joints, in both acute (single 10 mg CPPD intraarticular (IA), duration: 5 h) and chronic (repeated 10 mg CPPD IA, duration: 20 and 42 days) experiments, demonstrated a more intense histologic synovitis compared to cast immobilized knees (p = 0.0001). In chronic experiments, both CPPD injected and noninjected immobilized knees showed greater cartilage histopathologic-histochemical abnormalities (p less than 0.004) and significant reduction in cartilage hexosamine content (p less than 0.005), compared to exercised joints. CPPD injected knees, both exercised and immobilized, demonstrated an initial phase of increased cartilage biosynthetic activity (35S incorporation) at 20 days, compared to noninjected knees (p = 0.02), followed by a decline at 42 days (p less than 0.005). Our data indicate that joint movement enhances acute and chronic experimental CPPD crystal induced synovitis. Articular cartilage is more adversely affected by joint immobilization than by chronic crystalline inflammation. An optimum balance between exercise and rest seems necessary for patients with arthritis so that cartilage can be preserved but pain from active inflammation also controlled.

Is it mandatory to examine synovial fluids promptly after arthrocentesis?

Fifty synovial fluid (SF) samples from patients with various types of arthritis were examined promptly after joint aspiration and after storage at room temperature (22 degrees C) or at refrigerator temperature (4 degrees C) for 1 hour, 2 hours, 3 hours, 6 hours, 1 day, and 3 days, then weekly for 3 weeks and monthly for 2 months. We found that the leukocyte count (white blood cell [WBC] count) decreased within a few hours. In 4 SF samples from patients with mild inflammation (initial range 3,150-6,200 WBC/mm3), the WBC count decreased into a "noninflammatory" range (less than 2,000/mm3) within 5-6 hours. In 3 of 5 SF samples that on the first day were found to be laden with crystals of calcium pyrophosphate dihydrate (CPPD), the crystals were much less abundant and were difficult to recognize by the next day. CPPD crystals dissolved completely in all SF samples by 3-8 weeks of the study. Monosodium urate crystals remained detectable throughout the 8 weeks of study, but they became smaller, less birefringent, and less numerous with time. Clumps of apatite-like crystals persisted for several months. Most SF samples initially negative for apatite-like crystals remained negative over time. New, artifactual crystals, including alizarin red S-positive clumps or star-shaped arrays, plate-like structures, positively birefringent Maltese crosses, and hematoidin crystals, developed with time. Because of these observations, we urge prompt examination of SF specimens to avoid the problems of misdiagnosing borderline inflammatory fluids, missing CPPD crystals that dissolve with time, or over-interpreting the findings because of the new, artifactual crystals.