Compensated Polarized Light Microscopy

Compensated Polarized Light Microscopy Identification of Crystals in Synovial Fluids From Gout and Pseudogout Paulding Phelps, MD, A. Dean Steele, MD,...
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Compensated Polarized Light Microscopy Identification of Crystals in Synovial Fluids From Gout and Pseudogout Paulding Phelps, MD, A. Dean Steele, MD, and Daniel J. McCarty, Jr., MD

In

1961, McCarty and Hollander1 described the

presence of monosodium urate crystals in 15 synovial fluids from 18 acute gouty joints. Subsequently McCarty reported the presence of crystals in 141 of 150 fluids in acute gout. The examination of synovial fluid for urate crystals led to the discovery of a second type of crystal, calcium pyrophosphate dihydrate,2 in fluids from patients experiencing acute arthritis resembling gout. Named "pseudogout," this syndrome represents the clinical and pathologic elaboration of chondrocalcinosis as described roentgenologically by Zitnan and Sitaj.3 The intrasynovial injection of monosodium urate4,5 as well as a variety of other crystals into human and canine joints reproduces many of the clinical aspects of acute gout and pseudogout. The term "crystal induced synovitis" describes the nonspecific inflammatory reaction to microcrystalline irritants.

anesthetic might cause dissolution of crystals in a small sample of synovial fluid. The experienced phy¬ sician can usually enter a large joint, such as the knee, with less overall pain to the patient if local anesthetic is not used. Even the smallest amount of fluid obtained may be examined and should never be discarded, espe¬ cially if crystal-induced synovitis is suspected. De¬ finitive diagnosis can be made on a single tiny drop of fluid from the tip of the aspirating needle. With apparent failure to obtain joint fluid, a drop of blood from the aspirating needle may be found to contain crystals. A specimen sufficient to make a diagnosis can be obtained in the tip of a No. 26 nee¬ dle inserted into a small tophus, although a larger needle is obviously preferred.

Compensated polarized microscopy provides a rapid and accurate method for identifying monosodium urate and calcium

pyrophosphate crystals in synovial fluid, thereby confirming a diagnosis of gout or pseudogout. Failure to appreciate the diagnostic importance of accurate crystal identification in flu¬ ids removed from involved joints has been evident

in recent studies of gout.6 No complete description of all aspects of this technique exists in the medical literature. This com¬ munication describes ( 1 ) methods for obtaining and preparing samples of synovial fluid, (2) basic re¬ quirements for an inexpensive polarizing micro¬ scope, (3) a technique for examining synovial fluid, and (4) criteria for identification of monosodium urate, calcium pyrophosphate, and cholesterol

COMPENSATOR

crystals.

Obtaining Synovial Fluid Techniques for aspirating joints have been well described. Small, exquisitely tender joints usually require local anesthetic infiltration before insertion of a needle, and some mixture of anesthetic and spec¬

imen may occur. It should be remembered that urate crystals are water soluble and that a large volume of From the Rheumatology Research Laboratory, Philadelphia General Hospital, and the Rheumatology Section, Department of Medicine, Hahnemann Medical College and Hospital. Reprint requests to Second Floor\p=m-\Building25, 34th St and Curie Ave, Philadelphia 19104 (Dr. Phelps).

1. A

simple polarizing microscope with polarizer,

pensator, analyzer, and rotating stage.

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com¬

Disposable needles and disposable plastic aspi¬ rating syringes are preferred for fluid collection. These items are free of contaminating biréfringent material, which is not infrequently found in hospi¬ tal-washed test tubes, resterilized needles, and glass syringes. Only small volumes of heparin sodium (3 to 4 drops, 1:1,000 solution) should be used as an anti¬ coagulant for synovial fluid. The powdered antico¬ agulants such as oxalate are themselves crystalline; their use is confusing and may mask the presence of urate or pyrophosphate crystals.7 Preparation of Wet Smear The glass slide and coverslip are first thoroughly cleaned with lens paper. An amount of specimen is placed on the slide such that, when the coverslip is gently dropped onto the specimen without pressure, the expanding margin of the compressed fluid barely reaches the margin of the coverslip. The volume contained in a 1-mm bactériologie wire loop provides the proper amount, and

use

of the cleaned flamed

loop avoids contamination in transferring the speci¬ men to the slide. The coverslip is immediately rimmed with clear nail polish to retard specimen drying and subsequent precipitation of salts. To avoid contact between the polish and the objective lens, the slide should not be examined until the polish is dry. An ink marker line at the margin of the dried nail polish provides a means for identify¬ ing the proper microscopic focal plane. Bubble in¬ terfaces and small clots may also be used to identify the focal plane in specimens with few cells. It has recently been suggested that Wright's stained smears of the freshly aspirated fluid are easier to examine than wet

preparations." In our preparations were positive, crystals could be definitely identified in only

laboratory, when urate

wet

of 12 stained smears and pyrophosphate in 12 stained smears. Furthermore, crystal margins were indistinct, and an inordinate amount of extraneous material was present on the slide. Wet preparations are recommended for the beginner un¬ til more information is available. seven

only five of

Polarizing Microscope A polarizing microscope is recommended for rapid, accurate differentiation of urate and pyrophosphate crystals. Conversion of the ordinary biologic micro¬ scope with kits containing polarizer, analyzer, and compensator is awkward and may result in inade¬ quate optical resolution. The glass in the lenses of most

ordinary microscopes

may

cause

polarization

of light, and therefore, strain-free glass is used in the objectives of polarizing microscopes." The polarizing microscope ( Fig 1 ) used for crystal identification differs from the ordinary laboratory microscope in the following respects: (1) It con¬ tains two identical polarizing prisms or filters—the polarizer which is positioned below the condenser and the analyzer inserted at some point above the objective. In some instruments these are mounted

permit horizontal rota¬ tion of the polarizer and analyzer. (2) The me¬ chanical stage is mounted on a circular stage which rotates on a vertical axis ANALYZER coinciding with the center of the field. Objective lenses in polariscopes can be centered so that an ob¬ ject in the field of view re¬ mains in the center as the CRYSTAL stage is rotated. (3) A re¬ movable first-order red compensator is placed be¬ tween the objective and the analyzer. The firstorder red compensator is rolARIZER shown in the figure re¬ V\NV moved from its slot in the barrel of the microscope. The arrow showing the direction of vibration of the slower component of light in the compensator is mentally projected onto the stage as shown by the dotted lines. In practice, 2. Simplified concept of a crystal is centered in the polarized light (see text). field of view and the stage rotated so that the long axis of the crystal is parallel to this arrow. (4) All lenses and objectives should be of "strain free" (nonpolarizing) glass. Most microscope manufacturers sell relatively expensive pétrographie microscopes. However, relatively inexpensive instruments can be quite adequate. The total magnification provided by the objective x ocular should probably be 600 or to

^^^^^^^^^

•i^te^

^N\\\I

greater.

Polarized Light.—Detailed discussion of the op¬ tics can be found elsewhere'1 and the following is intended only as a simplified summary. Light traveling from the substage lamp toward the condenser is composed of elements having phases of vibration perpendicular to its direction of travel. The polarizing prism or filter passes light in only one plane as if through the slots in a grid. If only two perpendicular planes of light (A and B of Fig 2) are considered, it can be seen that only light in plane B will pass through the polarizer. This is because the "slots" in the grid of the polarizer run parallel to plane B, passing "light B" but not "light A." When the grids of the polarizer and analyzer are put at 90° to each other, the light vibrating in plane B, on reaching the analyzer, is now at right angles to the "grid" and no light from the source passes to the eye; thus, the microscopic field is dark. If an

optically anisotropic substance such as a sodium urate crystal is placed between the polarizer and the analyzer, the light passed by the polarizer will be rotated (here shown as an unlikely full 90°) enough by the crystal to pass through the grid of

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(

^

1 MONOCLINIC

2.TRICLINIC

3. CHOLESTEROL

D

/

v__) crystals and some calcium pyrophosphate crystals are needle shaped, or monoclinic (top); cal¬ cium pyrophosphate may appear as plates, or triclinic forms (middle); and cholesterol crystals are normally seen as brilliant notched plates (bottom). 3. Urate

the analyzer. Therefore, only that part of light B which has passed through the crystal will reach the eye, and the crystal will appear white on a black background with uncompensated polarized light. (It can be seen why the various glass lenses must not themselves "twist" the light). Compensated Polarized Light.—Definitive separa¬ tion of urate from calcium pyrophosphate crystals is accomplished by addition of the first-order red compensator to the optical system described above. This device separates light according to components of slow and fast vibration. It is inserted in the barrel of the microscope between the objective and the analyzer. The compensator retards red light so that the field background becomes red instead of black. (This mechanism is not shown in Fig 2, and the interested reader should consult a good pétrographie text for detailed explanation. ) The microscope should be set so that, by rotating the polarizer, the planes of vibration of the polarizer and analyzer at 90° to each other and at 45° to the direction of vibration of the slower compo¬ nent of light in the compensator. With the compen¬ sator removed, the darkest possible field is obtained by rotating the polarizer. The proper alignment when the compensator is replaced is then usually assured by the design of the microscope. This is not

always the

case and can be checked.9 Urate and calcium pyrophosphate may be both yellow and blue depending on the relation of the long axis of the crystal to the direction of vibration of the slower component of the compensator. The direction of vibration of the slower component is clearly marked on the compensator, either with the shorter of two arrows or with a single arrow marked gamma.9 It is shown in Fig 1 as a single arrow on the compensator, which has been removed from the barrel of the scope. For practical purposes, the axis of this line of slow vibration of the compensator is mentally pro¬ jected onto the stage (Fig 1) or aligned with the appropriate cross hair in the eyepiece. The stage is then rotated so that the long axis of the crystal is parallel to the axis of slow vibration of the com¬ pensator. If the crystal is blue in this position, it is calcium pyrophosphate and is called "positively biréfringent"; if it is yellow in this position, it is negatively biréfringent and is monosodium urate. When the calcium pyrophosphate crystal is then rotated 90° so that its long axis is perpendicular to the axis of slow vibration of the compensator, the crystal turns yellow while urate turns blue in the

comparable position.

Examination of the Slide for

Crystals

Sodium urate crystals (Fig 3, top, and 4, topleft and right) usually range in length from approxi¬ mately 2/1 to 10/t; crystals from tophi may be larger. Urates are brightly colored, ie, they are strongly biréfringent, and are usually seen as needles (Fig 3, top). Many intraleukocytic crystals are seen in joint fluid obtained during an acute gouty attack. Urates are more easily seen and identified than calcium pyrophosphate because of their larger size and stronger birefringence. Calcium pyrophosphate crystals (Fig 3, top and middle, and 4, bottom—left and right) range in length from 10/i down to the limits of optical defini¬ tion. As in acute gout, many intraleukocytic crystals are seen in the fluid from an involved joint. Their colors are usually, but not always, much less intense than urates, ie, they are weakly biréfringent. Occa¬ sionally the sign of birefringence may be difficult to determine, and in some specimens, the blue color can be elicited in only a few of the crystals. The crystals may be very weakly blue through only a few degrees of rotation when close to, but not exact¬ ly parallel to, the axis of slow vibration of the

compensator.

Both needle (Fig 3, top, and 4, bottom left) and plate forms (Fig 3, middle, and 4, bottom right) of

pyrophosphate crystals are frequently seen in the same specimen. In Fig 4, bottom right, the long axis of the smaller of the two crystals is almost parallel to the arrow, and the crystal is blue. Were the stage rotated 90 °, this crystal would turn yellow and the larger crystal would become blue. These crystals show weakly positive birefringence.

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Top left, Urate crystal in polymorphonuclear leuko¬ cyte. Axis of slow vibration of light in first-order red compensator is shown by arrow in all color plates (X2,940); top right, same crystal as in top left rotated

4.

Cholesterol

crystals appear as large, extremely or rectangular plate-like crystals often with a notch in the corner (Fig 3, bottom). Needle forms have been reported.'" Cholesterol crys¬ bright,

square

tals have been seen in the effusions of rheumatoid arthritis and in aspirates from extra-articular sites. Their pathophysiologic significance is unknown. Technique.—Several examining techniques may be helpful when crystals are not immediately seen but their presence strongly suspected.

1. It is suggested that the beginner make a smear of aspirated from a tophus and use this specimen as an empirical reference, realizing that urate crystals from

urates

tophus may be very large. 2. Carefully examine the leukocytes for intracellular crys¬ tals. Acute arthritis in gout or pseudogout probably does not occur without the presence of crystals within polymor¬ phonuclear leukocytes. a

90°

(x2,940); bottom left, calcium pyrophosphate crystal within a phagosome in polymorphonuclear leu¬ kocyte (X2.940); and bottom right, two triclinic calcium pyrophosphate crystals (x2,180).

3. Carefully examine small clots or unidentified debris in which crystals may be trapped. 4. Rack down the condenser to introduce more contrast (a partial "phase" effect). Extremely bright light sometimes obscures pyrophosphate crystals. 5. Scan the specimen under polarized light with the com¬ pensator removed. Weakly biréfringent pyrophosphate crys¬ tals will stand out strikingly against a dark background. Once located, a crystal can be identified by replacing the

compensator.

6. Phase accessories

are an

expensive addition, requiring

elaborate microscope but increasing its versatility. It may be of value to scan the slide using the phase objective.

a more

Contamination With Other Biréfringent Material. —Other biréfringent material may be present in the wet smear. Unclean glassware is a common source of such contaminants. Symmetry is a constant char¬ acteristic of urate and pyrophosphate crystals. Each has regularly parallel sides which are never jagged,

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rounded, or curved. "Broken" crystals may be sus¬ pected, but if a specimen contains crystals, many unbroken forms will be present. Contaminating material may be jagged, rounded, or irregular in shape and color. Corticosteroid crystals from re¬ cently injected joints may be confusing contami¬

crystalline anticoagulants. Scratches and coverslips should not be con¬ slides glass fused with crystals. Specimens should be examined within a few hours of the time they are removed from the patient. Minute specimens may be protected against drying by inserting the tip of the aspirating needle into a rubber cork during transit to the lab. nants

as are

on

Summary attack of arthritis suspected to be due to or pseudogout, the diagnosis can be rapidly gout and accurately established by identifying monoIn

an

sodium urate or calcium pyrophosphate crystals in the synovial fluid from an affected joint. Polarized light microscopy facilitates location of crystals, while the addition of a first-order red compensator to the system imparts characteristic color changes and allows definitive separation of urate from pyrophosphate crystals. Urates show strong negative birefringence, and pyrophosphates show weak posi¬ tive birefringence. A slide made from urates aspi¬ rated from a tophus provides a useful reference in learning to distinguish between these two crystal

types. Although conversion of the standard laboratory microscope is possible, a polarizing microscope with a rotating stage is recommended for crystal identi¬ fication.

This study was supported in part by grants from Merck Sharp & Dohme. the Eastern Pennsylvania Chapter of the Arthritis Foundation, and Public Health Service grant FR-00107.

References 1. McCarty, D.J., Jr., and Hollander. J.L.: Identification of Urate Crystals in Gouty Synovial Fluid, Ann Intern Med 54:452\x=req-\ 460 (March) 1961. 2. Kohn, N.N., et al: The Significance of Calcium Phosphate Crystals in the Synovial Fluid of Arthritic Patients: The "Pseudogout Syndrome": II. Identification of Crystals. Ann Intern Med 56:738-745 (May) 1962. 3. Zitnan, D., and Sitaj, D.: Chondrocalcinosis polyarticularis (familiaris): rentgenologicky a klinicky rozelor, Cesk Rentgen 14:27-34 (Feb) 1960. 4. Seegmiller, J.E.; Howell, R.R.: and Malawista. S.: The Inflammatory Reaction to Sodium Urate: Its Possible Relationship to the Genesis of Acute Gouty Arthritis, JAMA 180:469-475 (May 12) 1962. 5. Faires, J.S., and McCarty, D.J., Jr.: Acute Arthritis in Man and Dog After Intrasynovial Injection of Sodium Urate Crystals, Lancet 2:682-684 (Oct 6) 1962.

6. Wylie, J.D., and Steinbach, J.L.: Gout Associated With Calcification of Cartilage, New Eng J Med 275:745-749 (Oct 6) 1966. 7. Schumacher, J.R.: Intracellular Crystals in Synovial Fluid Anticoagulated With Oxalate, New Eng J Med 274:1372-1373 (June 16) 1966. 8. Good. A., and Frishette, A.W.: Crystals in Dried Smears of Synovial Fluid, JAMA 198:80-81 (Oct 3) 1966. 9. Chamot, E.M., and Mason, C.W.: Handbook of Chemical Microscopy, ed 2, New York: John Wiley & Sons, Inc., 1938 vol 1, pp 261-326. 10. Nye, W.H.R.; Terry, R.; and Rosenbaum, D.L.: Observation on Cholesterol Effusion in Rheumatoid Arthritis and in Idiopathic Cholesterol Pericarditis, abstracted, Arthritis Rheum 9: 528 (June) 1966.

FOR A SUCCESSFUL WRITER.-I do not know a training for a writer than to spend some years in the med¬ ical profession. I suppose that you can learn a good deal about human nature in a solicitor's office; but there on the whole you have to deal with men in full control of themselves. They lie perhaps as much as they lie to the doctor, but they lie more consistently, and it may be that for the solicitor it is not so necessary to know the truth. The inter¬ ests he deals with, besides, are usually material. He sees human na¬ ture from a specialized standpoint. But the doctor, especially the hospital doctor, sees it bare. Reticences can generally be undermined; very often there are none. Fear for the most part will shatter every defence; even vanity is unnerved by it. Most people have a furious itch to talk about themselves and are restrained only by the disinclin¬ ation of others to listen. Reserve is an artificial quality that is devel¬ oped in most of us but as the result of innumerable rebuffs. The doctor is discreet. It is his business to listen and no details are too intimate for his ears.—Maugham, W.S.: The Summing Up, New York: Doubleday, Doran & Co., Inc., 1938, pp 64.

FORMULA better

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