The identification of monosodium urate (MSU) and calcium pyrophosphate dihydrate (CPPD) crystals in synovial fluid or tissue biopsy samples is central to the definitive diagnosis of gout and CPPD deposition disease.
Basic calcium phosphate crystals are too small to be viewed with an optical microscope other than as crystal aggregates that appear as amorphous clumps.
Synovial fluid crystal analysis performed with a compensated polarized light microscopy is the gold standard for identifying MSU and CPPD crystals and for distinguishing these pathologic crystals from a variety of other particles present in the synovial fluid. However, this simple and rapid diagnostic approach is markedly underused in clinical practice.
The strong negative birefringence of MSU crystals and the weak positive birefringence of CPPD crystals, as well as differences in crystal shape under many, but not all, circumstances, helps distinguish MSU from CPPD crystals.
A rotating microscope stage is helpful to distinguish MSU and CPPD crystals from birefringent debris in the joint fluid. MSU and CPPD crystals have characteristic angles of extinction of birefringence that can be seen when rotated between angles parallel and perpendicular to the axis of slow vibration of light of the first-order red plate compensator.
The identification of monosodium urate (MSU) and calcium pyrophosphate dihydrate (CPPD) crystals is clinically relevant when performing a synovial fluid (SF) analysis for crystals. The search for apatite and other basic calcium phosphate (BCP) crystals remains of less certain clinical relevance at this point, but this issue is addressed in Chapter 22 . BCP crystals are too small to be seen with an optical microscope as anything more, at the most, as crystal aggregates in amorphous clumps. The occasional finding of other, rarer crystals plays only an anecdotal role. Hence, this review focuses on the search for MSU and CPPD crystals.
SF analysis for crystals has received little critical attention since its initial description, and the technique remains essentially unchanged. Crystal identification in SF is included in the core curricula in rheumatology, by both the American College of Rheumatology and the European Board of Rheumatology, UEMS Section of Rheumatology. SF analysis is a simple procedure requiring only a microscope fitted with polarized filters and a first-order red compensator; the identification of MSU and CPPD crystals provides an unequivocal diagnosis of gout and CPPD crystal arthritis. Using an adequately equipped microscope in the clinic allows an immediate and definitive diagnosis of crystal arthritis. There is no other immediate procedure that allows such an unequivocal etiologic diagnosis in any other arthritic disease.
Despite its immediacy and elegance, SF analysis is inconsistently implanted among rheumatologists, who as a community appear to have many that consider it dispensable. The reasons for this (other than the general low profile, in the past, of crystal arthritis) remain unclear, but as early as 1974 this lack of interest was noted. The authors have participated in the organization and tutoring of a good number of introductory workshops on crystal identification, including those held at the European League Against Rheumatism (EULAR) international congress meetings since 2002; key difficulties encountered among the attendants include (1) lack of familiarity with the use of the microscope, (2) the widespread concept that SF analysis is impossible without the fully equipped compensated polarized microscope, which is unavailable in many units, and (3) (for younger trainees) the lack of interest of their senior colleagues. In this chapter, we will review the current state of MSU and CPPD crystal identification—with a short review of other, rarer crystals—and specifically focus on practical aspects. We will also address some of the difficulties likely to be encountered by trainees and other less-experienced users. Currently at EULAR workshops, the possibility of detecting both crystal types—and even quite reasonably of distinguishing MSU from CPPD—by means of an ordinary microscope is being highlighted, as well as the availability of simple polarized microscopy at most pathology departments. By providing motivated trainees with such simple starting tools, we hope that they will feel motivated to start by themselves and, if successful, will be encouraged to perfect their technique and then to acquire the microscope necessary to comply with the standards.
MSU crystals were first seen in the 17th century by the inventor of the modern microscope (Leeuwenhoek), and their inflammatory potential in animal models had been noted by the end of the 19th century. MSU crystals were first identified in SF samples from gouty patients with acute arthritis with the use of a compensated polarized microscope by Daniel McCarty, Jr., after being noted with the use of an ordinary microscope by his mentor, Joseph Hollander. CPPD crystals were detected by McCarty while studying SF samples from patients with acute arthritis and presumed gout. The nature of both the MSU and CPPD crystals was confirmed by their x-ray diffraction pattern and by the capability of uricase to dissolve MSU crystals. However, their differing appearance and type of birefringence under the compensated polarized microscope allowed an easy and accurate distinction, and this microscope became the standard for crystal identification in SF.
MSU crystals in joint fluids have not been described outside of gout or some cases of asymptomatic hyperuricemia. Small numbers of CPPD crystals have been noted in the SF of patients with osteoarthritic joints and in other conditions such as the “Milwaukee shoulder syndrome” or in patients years after surgical knee meniscectomy ; the meaning of these findings remains undefined, and the identification of occasional CPPD crystals in a noninflammatory SF sample remains difficult to interpret. The presence of intracellular MSU and CPPD crystals in SF had been considered indicative of active joint inflammation of gout and pseudogout, but in SF drawn from asymptomatic joints, intracellular MSU and CPPD crystals are common in patients with gout and CPPD deposition disease. Of interest, the presence of either type of crystal in asymptomatic joints is associated with mild subclinical inflammation, as shown by a raised cellularity. Of particular clinical interest, when a joint containing either MSU or CPPD crystals is the seat of an infectious arthritis, the crystals will nevertheless be apparent in the SF analysis; if cultures are not obtained, the infection may easily be missed.
Crystal Identification, Validation Studies, and Quality Control
Real-life quality audits of SF analysis have been performed in Finland, Sydney (Australia), and different areas of the United States. SF samples were sent to laboratories considered to routinely perform SF analysis; the results were then compared to a gold standard, obtained from an experienced analyst. Both false-negative and false-positive results are common in all studies, and the rate of correct MSU crystal identification can be as low as 50% in some laboratories. These studies show the status of crystal analysis in real life and the results are worrisome. The studies were performed a few decades ago, but there is little to indicate that circumstances have changed. Different issues—methodological and otherwise—could partly account for these poor results. Observers were not identified; therefore, training and experience could not be evaluated, and in some laboratories, as many as 12 people participated in the analysis of a single SF sample. The microscopes that were used were not evaluated, and in numerous cases the technique might not have been as recommended, with no first-order red compensator available or with ordinary or simple polarized light inspection being omitted. So, even though the study results are poor, we are left wondering what would happen if current standards were applied and observers were appropriately trained. These surveys highlight the need to implement quality control programs in SF analysis rather than evaluate the reliability of the technique itself.
At least two other studies have been undertaken to evaluate the reliability of crystal detection. In the largest study to date, six observers with different experiences evaluated 143 unstained slides with MSU crystals, CPPD crystals, or no crystals. Results were unsatisfactory, with a low sensitivity for MSU detection and a moderate to low sensitivity and specificity for CPPD identification. Crystals were synthetically fabricated and then added to samples of SF obtained from patients. Data must be interpreted with caution as synthetic crystals can be larger and more irregular in shape and the larger crystals remain outside the cells and are not phagocytosed. A curious finding was that the rheumatologist with many years of experience in SF analysis had the lowest false-positive ratio but was most likely to miss crystals at low concentrations, suggesting a greater reluctance to report occasional particles or equivocal findings. In a recent study, we have shown that for trained observers, the detection and identification of natural crystals in fresh SF samples can be a consistent procedure. The training of the observers—four clinical analysis residents who were familiar with the ordinary microscope—consisted of a short course in crystal identification followed by a 3-month period of sample analysis. Then the participants examined 64 fresh SF samples containing MSU, CPPD, or no crystals. Sensitivities and specificities for both MSU and CPPD crystal identification were greater than 90%, although CPPD remained the more difficult crystal to identify.
Four microscope settings are useful in crystal analysis and in the training process: ordinary microscopy, simple polarized microscopy, compensated polarized microscopy ( Fig. 2-1 , A and B ), and phase microscopy. These four settings are usually achieved with the same microscope and different filters, except phase microscopy, which requires specific lenses and condenser. The basic laboratory microscope of most brands can adapt polarized filters for simple polarized microscopy, and many can also incorporate a first-order compensator (also known as retardation plate or lambda [λ] plate) to allow for compensated light microscopy. Many microscopes can also be fitted with phase lenses and condenser; phase microscopy is good to have, especially at the learning stage, but it is not necessary for crystal analysis.
Before starting training on crystal analysis, it is necessary to become acquainted with the use of the ordinary light microscope. The initial tool for crystal analysis was the geological polarized microscope, fitted with a rotating stage. It allows measurement of the angle of extinction of the birefringence (see later) and allows orientation of the crystals in relation to the axis of the compensator through rotation of the stage. These measurements are especially useful when there is a need to distinguish among a number of different crystal types, as a geologist may need to do, but in the joint we are essentially concerned with MSU or CPPD crystals, from which artifacts or the sporadic previously injected corticosteroid crystals are readily distinguished. In this context, corticosteroid crystals such as those of triamcinolone often have bright positive birefringence rather than the weak positive birefringence of CPPD. We believe that an ordinary microscope fitted with the appropriate lenses and filters is an adequate tool for crystal analysis.
To better understand our observations, the basic tool is described—the ordinary brightfield microscope—and then built-in complexity.
Brightfield microscopy: This is the illumination of conventional microscopes. Because SF preparations are fresh and unstained, detection of crystals with this illumination is based solely on shape. Cells and crystals are essentially transparent, so the height of the microscope condenser and aperture of the diaphragm must be regulated to obtain the best detail and contrast of the SF elements. Crystals are seen as regular structures with sharp edges, which can be found both inside and outside cells.
MSU crystals are always seen as thin needles of different sizes, intracellular or extracellular, and occasionally forming groups (with spherulites of MSU an infrequent finding). MSU crystals observed in SF from inflamed or uninflamed joints are smaller than those obtained by needling a tophus, which can be very large in comparison ( Fig. 2-2 ).
The polymorphic CPPD crystals may pose more difficulties. Observed under brightfield microscopy, typical crystals are rhombi or parallelepipeds. Some very thin and long parallelepipeds can look like rods or even needles; these are the only CPPD crystals that can be mistaken for MSU crystals. Often, CPPD crystals show a less regular shape, likely because of their position (it is a learning experience to observe typical crystals in a fresh preparation while the cells are still moving and watch the apparent shape of the crystal change with cell movement). In addition, some crystals may have broken off of larger crystals and therefore have a more irregular shape. Very small intracellular CPPD crystals are common and are seen as distinct inclusions that may have one or more linear borders or angles; tiny needles are also common. These smaller crystals are much better seen under a higher magnification with the ×1000 oil lens. The ×600 or ×630 lens (a good magnification level for crystal analysis) also shows them well. CPPD crystals are quite frequently seen inside a vacuole, while this appears not to be the case with MSU crystals ( Figs. 2-3, 2-4, A–C , 2-5, A , and 2-6, A ). The particularly inflammatory osmotic and membranolytic effects of MSU crystals discussed in Chapter 5 may account for this phenomenon.
The morphologic identification under ordinary light of CPPD crystals should be done only when enough typical crystals are seen; the finding of very small or less “typical”-looking crystals should prompt the observer to keep searching until he or she can identify an unquestionable crystal. In a small trial, both types of crystals were reasonably well distinguished with brightfield microscopy by their shape—63 SF samples (13 from patients with gout; 14, CPPD arthopathy; 1, both MSU and CPPD crystals; 35, different noncrystalline inflammatory arthritides) were blindly examined with brightfield microscopy by two different observers. The observers detected the presence of crystals in 100% (observer 1) and 97% (observer 2, who was a resident) of the samples; the crystals were properly identified with the brightfield microscope (in relation with the compensated polarized microscope) as MSU or CPPD in 92% (observer 1) and 87% (observer 2) of the occasions. In the sample containing MSU and CPPD crystals, both observers perceived the presence of both crystals.
Simple polarized light microscopy: Simple polarized light can identify crystals due to the intensity of their birefringence; it is often used to identify foreign material in pathologic specimens. Simple polarized microscopy is obtained when two polarized filters are added to the brightfield microscope. One of the filters is placed between the light source and the microscope slide (polarizer), while the second is placed above the lens (analyzer). A polarized filter is one that allows only the light vibrating in a plane parallel to its axis to pass through. In essence, you can imagine a polarized filter as a comb whose teeth are very closely packed and only the light vibrating parallel to its teeth is allowed to pass through. When the axes of the two polarized filters are placed perpendicular to each other, the light that passed through the polarizer is retained by the analyzer and the observer sees a dark microscope field (also known as crossed polarization). When the axes of the two filters are parallel to each other, observations can be made as with ordinary light. In an optic microscope, one of the filters is movable, allowing a change between brightfield and simple polarized light. The mechanism for crossing the filters is different depending on the microscope.
When a birefringent crystal is placed on the stage, the already polarized light beam that reaches it is split into two perpendicular components, none parallel to the incident ray, and with different velocities (fast ray and slow ray) and therefore out of phase with each other. The result is elliptically polarized light; the portion of this light vibrating parallel to the axis of the analyzer passes through it as plane polarized light, producing an image of a white crystal on a darkfield. In short, birefringent crystals seem to shine on a dark background because the microscope light can be seen through the crystal. By partially uncrossing one of the filters, less polarization is obtained; this allows the viewer to simultaneously distinguish the SF elements and the birefringence of crystals, which can occasionally be useful, especially for photography.
When seen under simple polarized light, MSU crystals shine brightly over the dark background. Brilliance, however, also depends on the intensity of the light source of the microscope—showing higher luminosity with the microscopes fitted with a stronger light source such as halogen lighting—and the height of the microscope condenser, which should be graduated to obtain the best image. Occasional MSU crystals may show no birefringence; this depends on crystal mass (very small crystals may be anisotropic) but also on the position of the axes of the crystal in relation with the axis of either the polarizer or the analyzer filters. When the crystal axis is positioned parallel to the axes of crossed polarized filters, birefringence disappears and the crystal is said to be in a position of extinction. On occasion, a crystal moving in the microscope field can fall in and out of the position of extinction and therefore show oscillating birefringence. Spherical aggregates of MSU crystals, all radiating from a center, have been reported, and incomplete radiating appearances are occasionally seen (an irregular radiating structure seen in a tophus is shown in Fig. 2-6 D–F ). After the viewer gains some experience, the bright MSU crystals are usually well detected at a magnification of ×200 with simple polarized microscopy. A better image is obtained at ×400; when searching for MSU crystals, there is rarely a need to use higher magnifications (see Fig. 2-2 , F–K ).
In the early descriptions of CPPD crystals, it was noted that under the simple polarized microscope they show only a weak birefringence, which is often absent. In a more recent trial, 10 SF samples from patients with CPPD crystal–proved acute arthritis were examined separately by two observers. First, a brightfield microscope was used; then, the same area was examined with a simple polarized microscope. Crystals showing any birefringence were annotated. Both observers found that less that 20% of CPPD crystals showed any birefringence. Ten SF samples from patients with crystal-proved gouty attacks were also examined; examination under simple polarized microscopy showed more crystals than observation by brightfield, indicating that some MSU crystals had passed unseen under the brightlight. Therefore, for CPPD, careful observation under brightfield appears to be the best method for detecting the crystals; when MSU is the suspected crystal, observation under simple polarized light is clearly better. It follows that before deciding that an SF sample has no crystals, it should be examined under both brightfield and simple polarized microscopy (see Figs. 2-4, D–F , 2-5, B , and 2-6, C ).
Compensated polarized microscopy: This method remains the current standard for crystal identification; it adds to the previously discussed system a first-order red compensator (λ or retardation plate), which helps to determine the amount of retardation in the wavelength of the compound ray emerging from the long dimension of the birefringent crystal. When this ray has a raised wavelength, it is viewed as yellow when parallel to the compensator axis (usually marked by a λ and an arrow) and as blue if perpendicular and the crystal is said to have negative birefringence or elongation. When the vibration of the slow ray is parallel to the long dimension of the crystal, it has a positive birefringence or elongation. Compensated polarized microscopy helps in the distinction between MSU and CPPD crystals, because the crystals have different birefringence: in MSU, it is negative (and bright) birefringence (see Fig. 2-2 , L and M ), while in CPPD, it is positive (and weak) birefringence (see Figs. 2-5, C–E , and 2-6, D and F ). The axis of the compensator can also be determined by keeping reference MSU crystals (a dried imprint of material needled from a tophi provides abundant MSU crystals for reference). Although it remains the standard tool for definitive crystal distinction, the crystal nature is already evident in most occasions based on the shape and intensity of birefringence under simple polarized light. Compensated polarized microscopy is most useful in the identification of very occasional needlelike artefacts or when MSU and CPPD crystals coincide in the same SF; this occurrence appears sporadic and no report on its clinical consequences has been published. A recent report examining SF from a patient with gout after cytocentrifugation also found a few CPPD crystals in patients with associated moderate to severe osteoarthritis, and possibly related to the osteoarthritis.
Phase contrast microscopy: This is a contrast-enhancing technique that produces high-contrast images of transparent specimens using ordinary light. When light waves that passed through the phase condenser pass through the preparation, some of its elements diffract and retard the phase (i.e., retard the wavelength). These retarded emerging waves are invisible to the naked eye but are transformed by the phase plate in the lens into amplitude differences observable at the oculars. Objects that modify the light phase—as do crystals—are seen lighter (see Fig. 2-6 , A2, B2, E, and G–K ). Phase microscopy was recommended by McCarty and shows the crystal’s shape particularly well; if available, phase microscopy and observation at ×1000 can be very good teaching instruments to familiarize a beginner with the different crystal shapes, particularly with CPPD crystals, including the very small intracellular ones. On occasion, it can also be useful for the expert analyst.
An ordinary microscope can be polarized by fitting it with polarized filters (obtainable online from commercial vendors) and cellophane tape can be used as a makeshift compensator. These are improvised polarized and compensated polarized microscopes, although the image quality may be inferior. Polarized filters can be cut by opticians from a large photographic polarized filter and adapted to the filter lodges of the microscope. Two filters are necessary—one under the stage, in the usual filter lodge of the microscope, and the other over it (most microscopes have a filter lodge in the inside of the piece that holds the lenses).