alizarin red s staining as a screening test to detect calcium compounds in synovial fluid

191

ALIZARIN RED S STAINING AS A SCREENING TEST TO DETECT CALCIUM COMPOUNDS

IN SYNOVIAL FLUID

HERNANDO PAUL, ANTONIO J. REGINATO, and H. RALPH SCHUMACHER

A simple, rapid screening method using alizarin red S stain and ordinary light microscopy to detect microcrystalline or noncrystalline calcium phosphate salts was used on wet drop preparations of synovial fluids. This proved to be helpful in detecting apatite crystal clumps and small calcium pyrophosphate dihydrate (CPPD) crystals missed by polarized light. The staining was positive in 100% of synovial fluids from patients later proven to have apatite and/or CPPD deposition diseases. Apatite and CPPD crystals were commonly found together in the same fluids. In addi- tion, some synovial fluids from patients with osteoarthritis, renal failure dialysis, rheumatoid arthritis, and gout also exhibited positive staining. The correlation of positive alizarin red S staining with radiologic evidence of osteoarthritis suggests that apatite crystals might be related to articular cartilage degeneration in different rheumatic diseases.

_ _ Prom the University of Pennsylvania School of Medicine

and Veterans Administration Medical Center, Philadelphia. Supported in part by grants from the Mariscal de Ayacucho

Foundation, Caracas, Venezuela, the Barsumian Fund, Philadel- phia, and the Eastern Pennsylvania Arthritis Foundation.

Hernando Paul, MD: Postdoctoral Research Fellow, Uni- versity of Pennsylvania School of Medicine and Veterans Adminis- tration Medical Center, Arthritis Section, and Assistant Professor of Medicine. Central University, Caracas, Venezuela; Antonio J. Keginato, MD: Associate Professor of Medicine, University of Pennsylvania School of Medicine and Veterans Administration Medical Center, Arthritis Section, and Associate Professor of Medicine, The Medical College of Pennsylvania, Philadelphia; H. Ralph Schumacher, MD: Professor of Medicine, University of Pennsylvania School of Medicine and Director. Arthritis-Immunolo- gy Center, Veterans Administration Medical Center.

Address reprint requests to Antonio J . Keginato. IMD, Associate Professor of Medicine, VA Medical Center, Arthritis Center (ISIK), University & Woodland Avenues, Philadelphia. PA 19104.

Submitted for publication October 8. 1981; accepted in revised form July I , 1982.

Study of synovial fluid by polarized light microscopy provides clinicians with a precise method of identifying most monosodium urate (MSU) and calcium pyrophosphate dihydrate (CPPD) crystals (1,2).

Recently, apatite crystals have been found in synovial fluid from patients with osteoarthritis (OA) and, more rarely, in patients with otherwise unexplained arthritis (3,4). However, individual apatite crystals are not identifiable under ordinary or polarized light because of their minute size (750-2,500 A in length) (5). Their precise identification requires electron microscopic techniques or x-ray diffraction analysis. A semiquantitative radioisotopic technique using 14C ethane-1 hydroxy 1, I diphosphonate (EHDP) binding to detect hydroxyapatite crystals in synovial fluid has been introduced by Halverson and McCarty (6,7). Although large amounts of apatite crystals tend to form aggregates which may appear as shiny but not birefringent clumps in synovial fluid, these shiny clumps are not always observed. At least some similar clumps might be due to protein OJ cell debris (4).

These observations had led us to search for simpler methods of apatite identification in joint effusions. This study reports the use of a calcium stain, alizarin red S, on wet drop preparations of synovial fluid as a screening test to detect apatite crystals. The alizarin tests were performed on synovial fluid from patients with a variety of different joint diseases. The presence of alizarin stained clumps correlated with the findings of apatite crystals by transmission electron microscopy (TEM) and with the radiologic grade of OA.

MATERIALS AND METHODS Alizarin red S staining techniques. Alizarin red S stain

(Harleco, Gibbstown, NJ), 2% solution in distilled water,

Arthritis and Rheumatism, Vol. 26, No. 2 (February 1983)

192 PAUL ET A L

Figure 1. Alizarin red S stain. Strongly positive test showing stained clumps in a small clot of osteoarthritis synovial fluid (ordinary light, original magnification x 100).

was adjusted to pH 4.2 by adding ammonium hydroxide drop by drop while stimng, using a Leeds and Northrup glass electrode pH meter (8). The solution was then filtered through a 0 . 4 5 ~ microfilter (Millipore Corporation, Bedford. MA), and kept in an amber bottle. This solution was refiltered through a 0 . 2 2 ~ microfilter immediately before use.

Routine synovial fluid analysis was performed on 275 joint effusions from 243 patients seen at the Philadelphia Veterans Administration Medical Center and Hospital of the University of Pennsylvania. Synovial effusions were aspirated into plastic disposable syringes and fluid samples were transferred to clean test tubes containing sodium heparin anticoagulant. Specimens were examined within 2 hours after arthrocentesis. Air exposure and freezing have been shown to increase the binding of I4C EHDP in synovial fluid that contains apatite crystals (7). The effects of prolonged air exposure, freezing, thawing, and dehydration of synovial fluid on alizarin red S staining were not studied in our specimens.

After a wet preparation was examined using a Zeiss compensated polarizing light microscope, one drop was aspirated from the bottom of the tube containing unspun synovial fluid. It was placed on a clean slide, mixed with a drop of 2% alizarin red S solution using the tip of a Pasteur pipette, covered with a clean cover glass, and observed under ordinary light microscopy within 3 minutes. Positive alizarin red S staining was graded according to the extent of heavy orange-red stained clumps observed per high power field under ordinary light microscopy. A test was considered strongly positive when the stained clumps were widely distributed and present in every field, or when many clumps were seen among the fibrils of small clots (Figure I ) . A wcak positive result was shown by scattered clumps among the fibrils; a negative test had no clumps in any field (Figure 2).

Sensitivity. The sensitivity of alizarin red S staining for apatite crystals was tested using different concentrations of synthetic apatite (Sigma Chemical Co., St. Louis, MO) or natural bone apatite (provided by Dr. C. Brighton, Orthope- dic Kesearch, Hospital of the University of Pennsylvania)

suspended in normal saline, synovial fluid, or serum. Apatite concentrations ranged from 0.002 pg to 10 mg/ml. Alizarin red S positive stained particles were seen with concentrations as low as 0.005 pg/ml. These results were comparable with those obtained by Dahl using a filter paper spot test and early calcified tissue sections, in which alizarin red stain revealed calcified tissue with a surface density of 0.02 to 0.002 pg/mm3 (9).

Specificity. Sodium heparin, synthetic MSU crystals (lo), synthetic cholesterol and corticosteroid crystals, alumi- num sulfate, magnesium chloride, sodium chloride (J.T. Baker Chemical Co., Phillipsburg, NJ), nuclear components (RNA, DNA), phospholipids (Applied Science Lab, State College, PA), collagen, fibrin, and mucopolysaccharides (provided by Dr. S. Jimenez, Connective Tissue Research Laboratory, University of Pennsylvania) suspended in normal saline or synovial fluid did not stain with alizarin red S.

Hydroxyapatite type 1, calcium phosphate type 11- brushite, calcium phosphate type I11 (Sigma Chemical Co.), /3-glycerophosphate, tetrasodium pyrophosphate, sodium phosphate, and calcium chloride (Fisher Scientific Co., Fair Lawn, NJ and J.T. Baker Chemical Co.) dissolved in normal saline or synovial fluid were stained very rapidly-within 10-15 seconds. Sites of calcium were covered by red stain and surrounded by an orange-red precipitate. Two patterns of precipitates were observed. Stained round or irregular precipitates of variable size (clumps) were seen in samples containing masses of tiny crystals as in hydroxyapatite type I and calcium phosphate type 111. The presence of similar stained clumps in synovial fluid was considered indicative of apatite crystal aggregates (Figure 1). Samples with larger crystals, such as CPPD, brushite, and oxalate, showed individually stained crystals with distinct crystal outlines (Figure 3).

Synthetic CPPD crystals developed the coloration within 3 minutes and usually exhibited weaker intensity (10). Natural calcium oxalate crystals ( 1 I ) stained after 30 minutes but their staining was most prominent only after 2 hours.

The findings of positive alizarin red S staining of crystals containing phosphorus, but not calcium or other divalent cations, was in disagreement with previous studies in which alizarin red S stain has been recognized as a

Figure 2. Alizarin red S stain. Negative test in an ostcodrthritis synovial fluid (ordinary light, original magnification x 10).

ALIZARIN RED S STAINING 193

A B Figure 3. Alizarin red S stain. A, Stained CPPD crystal (single arrow) and extracellular and intracellular stained clumps (double arrows), (ordinary light, original magnification x 60). B, Stained bipyramidal calcium oxalate crystal from a patient with renal failure undergoing long-term dialysis (ordinary light, original magnification x 60).

chelating stain for calcium, iron, lead, manganese, and barium (9,12). Alizarin red S is an anthraquinone derivative with 3 benzene rings, 2 oxygens, 2 hydroxyl groups, and a sulfonic radical in the form of a sodium salt. Cations are linked to alizarin red S by strong covalent linkages to oxygen and hydroxyl groups; this is believed to be the mechanism of the characteristic orange-red precipitate seen with calcium (9,12.13). The other cations listed above produce a distinctly different violet color. Other linkages can presumably be formed with the sulfonic chain and might account for the staining seen with some anions, such as phosphate.

Electron microscopic study. One hundred twenty-one synovial fluid samples were centrifuged for 20 minutes at 2,500 revolutions per minute (rpm), and pellets were fixed, within 120 minutes of arthrocentesis, in half-strength Kar- novsky's glutaraldehyde paraformaldehyde fixative. After 15 minutes' fixation, the pellets were minced in % x '/z mm pieces, fixed for 4 more hours at room temperature, and processed by a previously reported technique (14).

Microprobe analysis. Thin sections on copper grids from 31 synovial fluid specimens prepared as above were carbon coated and examined unstained on a Zeiss EM 10 TEM with a lithium-drifted silicon energy dispersive x-ray detector (Kevex) interfaced with a multichannel analyzer (Kevex 7000) and a computer system. Crystals were ana- lyzed using a spot size of 0 . 7 5 ~ diameter, and 200 seconds analysis time.

X-ray diffraction analysis. Chalky material aspirated from the joints of 4 patients, and 7 synovial fluid sediments were studied by x-ray diffraction analysis. Approximately 5 cc of each synovial fluid sediment was treated with 2-3 drops of hyaluronidase solution containing 150 turbidity reducing units per ml (Wydase, Wyeth Lab, Inc., Philadelphia, PA) for 15 minutes at 20°C. After centrifugation for 20 minutes at 2,500 rpm, 5 cc of 1% solution of 1 : 250 trypsin (Difco Lab, Detroit, MI) in deionized water was added to the residue, and this was incubated for 4 hours at 37°C (14). The sediment

was washed twice in saline and centrifuged at 2,500 rpm for 10 minutes. The resulting synovial fluid sediments and the chalky materials were dried in an oven at 130°C for '/r hour, and studied by x-ray diffraction. A Debbye-Scherrer powder camera (North American Phillips) of 114.6 mm diameter using chromium K alpha radiation with a vanadium filter was $lized. Measurements were based on wave length 2.29092 A. The specimens were exposed for 20 hours at 60 kV and 20 mamp.

Diagnostic criteria. The following diagnostic criteria were applied for patients whose synovial fluid was studied.

Apatite deposition disease was diagnosed by the presence of otherwise unexplained arthritis or periarthritis with radiographic evidence of synovial membrane and per- iarticular calcific deposits. All patients had apatite crystals in their synovial fluid confirmed by x-ray diffraction analysis. Patients with any other coexisting crystals and osteoarthritis were excluded for the purpose of this study.

CPPD deposition disease was diagnosed by the presence of weakly positive birefringent rod or rhomboidal shaped crystals in the synovial fluid (56 patients). Koentgen- ographically typical chondrocalcinosis was seen in 27 patients (IS). Patients whose synovial fluid had CPPD crystals, and who also had another associated joint disease, were listed as having CPPD deposition disease only. They were excluded from other diagnostic groups for this study. Thus, 5 synovial fluids with both CPPD and MSU crystals, 1 with CPPD from a patient with rheumatoid arthritis (RA), and 25 with CPPD from patients with OA were listed only as CPPD disease in this study.

Osteoarthritis was diagnosed on the basis of nonin- flammatory joint fluid (synovial fluid cell count <2,000). with roentgenographic evidence of cartilage degeneration and/or bone remodeling in the absence of criteria for any of the other conditions studied.

The diagnosis of gout was based on the demonstration in the synovial fluid of negatively birefringent rod- or

194 PAUL ET AL

needle-shaped crystals typical of MSU crystals, by compensated polarized light.

Patients with a total episode of acute arthritis of less than 1 month’s duration, who did not fulfill diagnostic criteria for any accepted disease entity, were classified as having undiagnosed acute synovitis. Undiagnosed chronic synovitis was designated in those patients with persistent or recurrent joint disease of more than 1 month’s duration, still eluding any standard disease classification.

Internal derangement of the knee was diagnosed by the presence of characteristic clinical and arthrographic findings (16). Patients with symptomatic joint effusions while in a long-term dialysis program were included as a separate diagnostic group, because no other explanation was obtained for their arthritis. Diagnoses of septic arthritis, RA, systemic lupus erythematosus, and ankylosing spondylitis were made using the American Rheumatism Association or other well accepted criteria (17-20).

Radiologic criteria. Roentgenograms of 134 joints from which synovial fluid samples were obtained were read “blindly” by two of the authors. They were graded on a 0- IV scale, looking for OA changes using the method of Kellgren and Lawrence (21) as follows:

Grade 0 (none): Absence of roentgenographic changes of OA.

Grade I (doubtful): Asymmetric narrowing of joint cartilage associated or not associated with sclerosis of subchondral bone.

Grade I1 (minimal): Narrowing of joint cartilage associatcd with subchondral sclerosis, and small ostco- phytes on the joint margin or on the tibia1 spines.

Grade 111 (moderate): Increased narrowing of joint

cartilage, subchondral sclerosis, larger osteophytes, and pseudocystic areas with sclerotic walls in the subchondral bone.

Grade IV (severe): Same as Grade 111, but with subluxation and gross deformity of the bone ends.

RESULTS Alizarin red S staining in synovial fluid. Table 1

summarizes the alizarin staining of clumps in 275 synovial fluid samples in the different diagnostic groups. Stained CPPD or other large crystals are not included in this table.

Apatite deposition diseuse. All 6 synovial fluids in this group showed strongly positive tests with stained clumps. The clumps ranged in size from 3p to 20p. They were often seen widely distributed in the slide, and exhibited similar intensity of stain and shape to those observed in samples of synthetic apatite crystals. TEM studies in all 6 fluids showed abundant small, needle-shaped crystals, identified as apatite-like crystals (Figure 4).

CPPD deposition diseuse. By definition, the 56 synovial fluids in this group containcd CPPD crystals. In all fluids, not only stained CPPD but also stained clumps were observed by light microscopy. The clumps were more often seen among fibrils of small

Table 1. Results of alizarin red S staining in 275 synovial fluid studies

Alizarin red S: grade of positivity of staining of clumps

Strong positive Weak positive Total positive Total fluids

n* % n 9i n % n

Apatite deposition disease

CPPD deposition diseaset

Dialysis Osteoarthritis Gout Rheumatoid arthritis Systemic lupus ery-

thematosus Undiagnosed chron-

ic synovitis Seronegative spon-

d ylarthropathy Septic arthritis Undiagnosed acute

synovitis

6

50 2

30 6 9

0

1

0 I

0

I00

89.28 20 37.04 IS 26.47

0

8.33

0 8.33

0

0 0 6

6 10.71 56 4 40 6

22 27.16 52 8 20 14 2 5.38 I I

I 25 I

I 8.33 2

I 1 4 . 2 ~ I 0 0 I

0 0 0

I00

I00 60 64.20 35 32.35

2s

16.66

14.38 8.33

0

6

56 I0 81 40 34

4

12

7 12

10 Internal derange-

ment of knee 0 0 0 0 0 0 3

* n = number of patients. + CPPD = calcium pyrophosphate dihydrate.


Figure 4. Hydroxyapatite crystals from a patient with apatite deposition disease. Stained clumps had been previously observed with alizarin red S (transmission electron microscopy. original magnification x 16,000).

clots and rarely seen widely distributed in the slide. Fifty fluids (89.29%) were strongly positive for clumps, and 6 (10.71%) weakly positive. Of those 50 synovial fluids exhibiting a strongly positive result. 33 were studied by TEM. Apatite-like crystals only were found in 8 , apatite-like plus CPPD-like crystals in 18, and CPPD-like crystals only in 5. In 2 fluids neither apatite-like nor CPPD-like crystals were observed. Of the 6 fluids with weakly positive staining for clumps, only 1 was studied by TEM. CPPD-like crystals, but not apatite, were seen in that specimen. There were no significant differences in the amount of alimrin red S stained clumps or apatite-like crystals observed in patients with CPPI) dcposition disease and chondrocalcinosis, compared with patients having roentgenographic evidence of only degenerative arthritis and no articular cartilage calcification.

Osteoarthritis. Of 81 OA synovial fluid samples without CPPD crystals seen by polarized light, 52

showed alizarin stained clumps. In 30 (37.04%), the staining was strongly positive. TEM studies were performed on 15 of these 30 fluids. Apatite-like crystals only were found in 7, apatite-like plus CPPD-like crystals in 3, and no crystals in 5. Of 22 fluids with weakly positive results, 10 were studied by TEM. Apatite-like crystals only were found in 3. apatite-like plus CPPD-like crystals in 1, CPPD-like crystals only in 2 , and no crystals in 4. TEM studies performed on 14 synovial fluids of 29 with a negative alizarin test showed neither apatite nor CPPD crystals. Six fluid samples showed small CPPD-like crystals of about 3- 5p stained by alizarin red S that had not been seen with Compensated polarized light.

Miscellaneous. Nine of 10 synovial fluid samples obtained from patients with symptomatic knee effusions while in a long-term dialysis program exhibited alizarin stained clumps. They were strongly positive in 2. Three also showed alizarin red S positively

196 PAUL ET AL

bipyramidalO.5-3Op crystals characteristic of calcium oxalate (Figure 3B). TEM and crystallographic studies of these 3 patients have previously been reported by Hoffman et a1 (1 1). Stained clumps were also seen in 14 of 40 gouty synovial fluids (6 strongly positive), and in 11 of 34 fluids from patients with RA (9 strongly positive). TEM studies were performed in 13 of those synovial fluid samples which were alizarin red S strongly positive. Apatite-like crystals only were found in 6, CPPD-like plus apatite-like crystals in 3, CPPD-like crystals only in 3, and no crystals in 1. TEM studies in 4 synovial fluid specimens that were alizarin red S weakly positive and in 9 that were alizarin negative showed neither apatite-like nor CPPD-like crystals .

As seen in Table 1, most of the synovial fluid samples from patients with systemic lupus erythematosus, septic arthritis, seronegative spondylarthropath- ies, undiagnosed synovitis, and internal derangement of the knee did not stain with alizarin red S.

Apatite and CPPD crystal identification criteria by TEM. Apatite-like crystals (Figure 4) were rod- or

needle-shaped, and uniformly dense or with less dense centers. They were 100-750 A in width and up to 4 times as long (4). Most crystals were densely packed in clumps but some were loosely scattered. CPPD-like crystals (Figure 5) appeared generally rhomboidal or rod-shaped and were much larger and thicker than apatite crystals. They were electron dense at the beginning of exposure to the electron beam, but within a few seconds many electron lucent areas developed and the crystals exhibited a foamy appearance (14).

Correlation between alizarin red S and TEM findings. Table 2 shows the overall correlation between grades of alizarin red S staining and crystal features by TEM studies in 121 synovial fluid samples, irrespective of the clinical diagnosis. It was found that the higher the intensity of alizarin positive tests for clumps, the higher the possibility of finding apatite-like crystals by TEM.

In the 70 fluid specimens with strongly positive tests for clumps, apatite-like crystals were found by TEM in 54 (77.14%) and apatite-like and/or CPPD-like crystals in 62 (88.57%). Only in 8 samples ( I 1.43%)

6

A B Figure 5. A, Tiny crystals from a synovial fluid in which no crystals had been observed by polarized light microscopy. Although the alizarin red S stain had been strongly positive only for clumps, one crystal has the typical configuration of CPPD. The irregular one is not typical and might be some other calcium salt (transmission electron microscopy, original magnification x 31,500). B, CPPD crystals from fluid in which CPPD had been detected using polarized light microscopy (transmission electron microscopy, original magnification X 16,000).


Table 2. transmission electron microscopy (TEM) crystal findings in 121 synovial fluids

Correlation between intensity of positive staining of clumps with alizarin red S and

Alizarin red S Strong Weak

TEM features positive positive Negative

Apatite-like only 27 (38.57%) 3 (18.75%) 0 (0%)

CPPD-like* 27 (38.57%) I(6.25%) 0 (0%) CPPD-like only 8 ( I I .43%) 3 (18.75%) 0 (0%) Negative 8 (11.43%) 9 (56.25%) 35 (100%)

‘Total 70 16 32

Apatite-like and

* CPPD = calcium pyrophosphate dihydrate.

were neither apatite nor CPPD found by TEM, although in 2 of them CPPD crystals had previously been identified by polarized light.

In 16 synovial fluids with clumps but weakly positive tests, apatite-like crystals were found in 4 (25%), apatite-like and/or CPPD-like crystals in 7 (43.75%), and no crystals were found in 9 (56.25%).

All 35 synovial fluids with negative alizarin tests were negative for CPPD or apatite by TEM.

Two observations should be made regarding the findings shown in Table 2. In 10 synovial fluids in which CPPD crystals had been observed by polarized light, such crystals were not found by TEM. In 8 of them, only apatite-like crystals were found and in 2, neither CPPD nor apatite crystals. In 12 fluids with a positive alizarin test but without CPPD crystals identified by polarized light, small CPPD-like crystals were found by TEM (Figure 5).

Microprobe and x-ray diffraction analysis. For- ty-two alizarin red S positive specimens containing apatite-like crystals by TEM were studied by microprobe or x-ray diffraction analysis.

Thirty-one specimens were studied by microprobe analysis. In 28 of them, masses of tiny apatite- like crystals exhibited a C d P ratio of about I .5-1.7: 1, as should be expected with hydroxyapatite. In the remaining 3 specimens the clumps containing apatite- like crystals were so small that they did not seem to give off sufficient x-rays for elemental analysis.

Eleven specimens studied by x-ray diffraction analysis confirmed the presence of hydroxyapatite in 9 cases.

Alizarin red S staining and radiographic signs of osteoarthritis. The correlation between grade of positive alizarin tests and radiologic grade of OA in 134 patients, regardless of clinical diagnosis, is summa- rized in Figure 6. Of 59 patients with strongly positive tests, 37 (62.71%) had radiologic grade 111 or IV OA;

grade I1 or higher OA was found in 55 patients (93.22%). Only 4 patients had grade I or 0; 3 of them had CPPD crystals on the fresh smear, and the other had apatite deposition disease.

Of 17 patients with weakly positive tests, OA

0. 00. 00. 00. N/ 0 .

. O i 0 0 0 0 0 0 0 . 0 0 0 0 0 0 0 .

0 . 0 0 0

1 1 I $ 0

0

. . 0 0 0 0 0 0 0 0

0 0

0

3 0 0 0

00 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

0 0 0 0 0 0 0 0

. 0: 0 0 0 0 0

0.

I STRONG . WEAK NEGATIVE POSITIVE POSlTNE

Alizarin Red S

Figure 6. Correlation between intensity of alizarin red S (tests with stained clumps) and radiologic gradc of osteoarthritis in 134 patients. 0 = with CPPD, 0 = without CPPD.

198 PAUL ET AL

grade I1 was found in 9 (53%), and 8 patients (47%) were grade I or 0. No patients were grade 111 or IV.

In 58 patients with negative alizarin tests, only 13 (22.41%) were found to have a radiologic grade 11 of OA, while 45 patients (77.59%) had grade I or 0. No patient was grade 111 or IV.

DISCUSSION

The difficulties in detection of apatite crystals on wet drop preparatiohs of synovial fluid led us to investigate alizarin red S as a rapid screening test which could offer another diagnostic aid when calcium phosphate crystal deposition diseases were suspected. Alizarin red S has been widely used as a reagent for calcium staining of bone since 1905 (22). The solution is stable, but must be maintained at pH 4.2 for better color contrast and uniform staining (8,13). The reagent must be filtered before using to avoid stain precipita- tion. Sites of calcium are covered and surrounded by an orange-red precipitate, easily distinguished from the paler orange background.

After testing several different amorphous and crystalline calcium salts used as positive controls, it became clear that the reagent was very effective in staining every one of them. All calcium and calcium phosphate compounds rapidly developed the bright orange-red color, although CPPD crystals showed paler stain (Figure 3). Even calcium oxalate crystals, which have been reported not to be stained by alizarin red S (23), developed the orange-red color after 30 minutes. It is important, however, to also note that the stain is not specific for calcium, since sodium phosphate and tetrasodium pyrophosphate gave a similar orange-red precipitate.

The theory that alizarin red S has an affinity for calcium phosphate compounds in synovial fluid was supported by the finding of stained clumps in all 6 patients with apatite deposition disease and by stained CPPD crystals and clumps in all 56 patients with CPPD deposition disease (Table 1). Because the stained clumps were usually found among fibrils in small clots, compounds normally present in synovial fluid were tested with alizarin red S. Purified RNA, DNA, phospholipids, and mucopolysaccharides suspended in normal saline or synovial fluid showed negative stainings, suggesting that phosphate included in some of these compounds was not responsible for the positive staining observed in the tested synovial fluid. The theory

that the alizarin red S stained clumps might represent apatite aggregates was further supported by the results shown in Table 2. This shows that the possibility of finding apatite-like crystals by TEM was much higher in those fluids with strongly positive tests (77%) than in those with weakly positive tests (25%), while no apatite was found in synovial fluid with negative staining. Crystals were further confirmed to be apatite by x-ray diffraction and microprobe analysis in 37 specimens in which such studies were possible. Those 8 synovial fluid samples with strongly positive tests, in which apatite was not found by TEM, might represent either a false negative by TEM or a false positive reading for apatite with alizarin red S. We found that 10 synovial fluid samples with CPPD crystals shown by polarized light were false negative by TEM. The same might have occurred with apatite crystals since only limited electron microscopy studies (i.e., exami- nation of 1 grid) were performed on each specimen due to time and cost constraints.

That apatite was the only compound responsible for all alizarin red S stained clumps in the synovial fluid is, however, questionable. Other calcium phosphate compounds or tiny CPPD crystals might also have been responsible for the stained clumps. From existing knowledge of the chemistry in in vitro formation of CPPD and apatite crystals, one could speculate that intermediate crystalline or noncrystalline calcium salts other than the final product might be seen in the synovial fluid of patients with CPPD deposition, apatite deposition, or osteoarthritis (24). This idea is further supported by reports of mixed crystal deposition in patients with CPPD disease and osteoarthritis (25-27). These findings might also, of course, be of potential clinical and pathogenetic qignificance and require further study.

The finding of stained clumps in 100% of synovial fluid samples from patients with CPPD crystals suggests that apatite was associated with CPPD in every case. However, this was only incompletely supported by TEM, in which 76.47% of 34 synovial fluids of CPPD deposition disease showed the presence of apatite-like crystals. There were thus 23.53% of those fluids with stained clumps in which apatite was not observed. These fluids might either represent a false negative by TEM, or suggest that the stained clumps were produced by amorphous calcium phosphate or by aggregation of tiny CPPD crystals. Tiny CPPD crystals so small that they were recogniLed only by TEM have been described by Honig et al ( 2 8 ) .


Dieppe et a1 have also observed that these crystals are often only seen in clumps of fibrin (26). In our study, tiny CPPD-like crystals seem to have been at least in part responsible for the stained clumps in 12 synovial fluid specimens, in which CPPD was not identified by polarized light but was later recognized by TEM. Crystals termed CPPD-like by TEM in this study are ultrastructurally typical of CPPD but might also in- clude some other calcium salts (such as brushite), which might not be distinguishable by TEM. Electron probe and x-ray diffraction were not performed on these rare and small crystals.

Hydroxyapatite crystals have been identified in this report (Table 1) and in previous studies in patients with OA (3,4,6,7), on long-term dialysis, and even in RA (27). To the best of our knowledge, however, these crystals have not been reported associated with gouty synovial fluid. Our findings of stained clumps in gouty synovial fluid, however, should not be unexpected. Tophi have the tendency to become calcified and apatite has been found in such tissues (29); if release of MSU crystals (from a calcified tophus or a tophus eroding bone) into the synovial fluid occurred, then one might expect to find not only MSU, but also apatite crystals in such synovial fluid.

The nonspecific finding of apatite crystals in many chronic joint diseases led us to correlate the alizarin red S stain findings with the radiographic grade of OA. The high correlation found in our study between the intensity of positive alizarin red S stained clumps and OA suggests that apatite crystals are commonly associated with articular cartilage degeneration regardless of the clinical diagnosis. Halverson and McCarty, using a radioactive diphosphonate binding technique to detect apatite in synovial fluid, found binding material suggestive of apatite in 28% of nonin- flammatory synovial fluids (6). The binding material in their study correlated with the presence of CPPD crystals and with roentgenographic evidence of cartilage degeneration.

On the basis of our observations we conclude that alizarin red S staining of fresh wet preparations of synovial fluid is a rapid, simple, and sensitive technique, helpful in indicating the presence of apatite crystals and other calcium phosphate compounds. Although the stain is not specific, it was of value as a screening test before using electron microscopic techniques or x-ray diffraction analysis to confirm the presence of apatite crystals. Since apatite is usually not birefringent, it is helpful to differentiate it from

other particles such as fat globules. cell detritus, and artefacts. The staining also helps distinguish tiny CPPD or oxalatc crystals from non-calcium-containing crystals such as depot corticosteroids. Using a centrifuged synovial fluid washed pellet followed by trypsin digestion might increase the specificity of the test, as has been the case with the I4C EHDP binding assay to detect putative apatite crystals (7).

The coexistence of apatite and CPPD crystals in a high percentage of the synovial fluid samples studied suggests that the association of these crystals might be higher than that reported previously by other investi- gators (30). The relative role of each crystal is not yet elucidated.

ACKNOWLEDGMENTS The authors acknowledge the superb technical assist-

ance of Gilda Clayburne, Susan Rothfuss, and Marie Sieck and the secretarial help of Esther Lobb and Joanne Logui- dice in preparation of the manuscript.

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alizarin red s staining as a screening test to detect calcium compounds in synovial fluid

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