EULAR on-line course on Rheumatic Diseases

Crystal arthropathies

Leonardo Punzi, Edward Roddy, Priyanka Chandratre

A previous version was co-authored by Franco Schiavon, Eliseo Pascual, Thomas Bardin
and Pascal Richette

IN-DEPTH DISCUSSION I

Essentials of crystal detection and identification in

synovial fluid
 Crystal arthropathies– Module 12

Identification of monosodium urate (MSU) crystals and calcium pyrophosphate (CPP) crystals provides an
aetiological definitive diagnosis of gout and CPP crystal arthritis. The diagnosis of these two conditions is often
approached clinically, and when the disease has a ‘typical’ presentation (such as repeated podagra in a
hyperuricaemic patient or an acute knee arthritis in a patient with ‘typical’ chondrocalcinosis) the diagnosis is
usually correct. But as the presentation diverges from the most characteristic one and in the absence of
synovial fluid (SF) analysis for crystals the possibility of error increases, and requires a diagnostic investigation
which is not always simple or conclusive. It is in such patients where the diagnosis is less evident that other
doctors usually consult rheumatologists, and they expect—and know we can provide—an evidence-based
diagnosis for crystal arthritis. Other crystals visible by the optic microscope are of limited interest. This subject
is dealt with in greater depth in the EULAR online course.

The size of MSU and CPP crystals allows detection and reasonable provisional identification with an ordinary
optical microscope (Pascual et al, 1989). Fitting the microscope with polarised filters—analyser and polariser—
(simple polarised microscope) allows detection/identification by shape + birefringence; this microscope is
commonly used in pathology departments. Finally, adding a first-order red compensator to the system
(compensated polarised microscope) allows more definitive identification of both crystals; this last setting
remains the standard for crystal identification in SF (Phelps et al, 1968). After training, crystal identification in
SF is consistent (Lumbreras et al, 2005). Beginners fear they may have difficulty in differentiating MSU and CPP
crystals but, in general, most analysts find the appearance of the crystals is different and recognise them at
first glance. Training consists in becoming familiar with both crystals and then recognising them in new
samples. An ordinary microscope fitted with the proper filters (some most popular brands provide sets of
these) is the correct tool. A ×600 lens allows better vision and, for learning, a ×1000 lens allows a closer look
(especially important in the polymorphic CPP crystal) and familiarisation with the crystals. Finally, a bright
halogen light or similar is important for observation under polarised light—especially simple polarised light—
since the strength of the brilliance of the crystal depends on it. Beginners should first become familiar with the
use of the microscope—focusing, regulating the condenser and so on—and also with other elements in SF
such as cells and other occasional common artefacts—for example, cotton fibres, starch from gloves or dust
particles.

Both types of crystals are seen in SF taken from joints affected by an acute attack and in gout also in SF from
previously inflamed joints if the patients have not received urate-lowering treatment. CPP crystals have also
been regularly identified in SF samples from previously inflamed joints—a treatment which can eliminate the
crystals from the joints is much needed. In gout, crystals are regularly recovered by needling a tophus.

1. With an ordinary microscope both MSU and CPP crystals are easily seen and with experience can be
identified by their shape, and can be used when polarising microscopes are unavailable.

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(i) All MSU crystals are needle shaped, their size varying from very small to about twice the diameter
of a white blood cell both intracellularly and extracellularly (figures 1A, B). Often crystals are abundant
and are easily seen already in the first ×400 microscope field examined; when scarce, detection of the
crystals with this method may be problematic.

(ii) CPP crystals are polymorphic. Their shape varies from a rhombus to thin needles; rods or thick
needles and parallelepipeds are intermediate forms (figures 2A–D, 3A, C). Sizes vary from very small to
about twice the diameter of the usual white blood cell. Extracellular crystals are seen but it is worth
checking for crystals inside cells where they are very commonly found. Here using a magnification of
×1000 has an advantage in enabling acquaintance with the crystals, facilitating later recognition.
Examination with the ordinary light microscope allows a reasonable detection and identification of
MSU and CPP crystals and it is worth doing if no polarised microscope is available.

Figure 1: Bright field microscopy. (A) ×400; (B) ×1000 monosodium urate (MSU) crystal. MSU crystals are well
distinguished and all crystals are needle shaped. If abundant (and often they are) their presence is
immediately obvious.

A B

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Figure 2: Bright field microscopy. (A, B) ×400; (C, D) ×1000. (A) Abundant calcium pyrophosphate (CPP)
crystals; (B) single parallelepiped crystal. (C, D) To become familiar with the crystals ×1000 magnification
offers greater detail. Photomicroscopy offers only a very thin plane on focus. At the microscope the fine-
focus knob allows all the structure to be brought into focus and seen more clearly.

A B

C D

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 Crystal arthropathies– Module 12

Figure 3: (A, C) Bright field microscopy ×600. Calcium pyrophosphate (CPP) crystals are seen and
distinguished by shape. (B, D) Same as A and C after crossing the polarised filters for simple polarised
microscopy. (E) Simple polarised microscopy ×1000. These figures show the range of birefringence of CPP
crystals from none to quite brilliant—seldom reaching that of monosodium urate crystals. About 80% of CPP
crystals do not show any birefringence. (For photography, polarised filters are not totally crossed to allow
some background detail.)

A B

C D

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 Crystal arthropathies– Module 12

2. The simple polarised microscope allows detection and identification by birefringence.

(i) All MSU crystals are strongly birefringent and shine brightly against the dark background of the
microscope field (which results from crossing the polarised filters) (figures 4A–C). This setting is
the ideal for finding MSU crystals if they are few, since owing to their brightness they are easily
seen in the dark microscope field. Small MSU crystals may not show birefringence. Additionally,
birefringence cannot be seen when the long axis of a crystal is oriented parallel to the axis of
either of the polarised filters (position of extinction).
(ii) Only about one-fifth of CPP crystals show any birefringence (Ivorra et al, 1999) and, in general, it
is much fainter than that of MSU crystals. Therefore, absence of birefringence should not be taken
as an indicator of the absence of CPP crystals (figures 3A–E). The often very thin rods or even
needle-shaped CPP crystals usually show no birefringence, allowing easy distinction from MSU
crystals.

3. Compensated polarised microscopy is the standard tool for crystal analysis in SF. It determines the sign of
birefringence, which depends on whether the wavelength traversing the long axis of the crystal has increased
or reduced when polarised light traverses them.

(i) MSU crystals show negative birefringence, or elongation, recognised because the long axis of the
crystal is yellow when parallel to the compensator axis (or direction of slow vibration)—most
often marked with an arrow and the Greek letter λ, and blue if perpendicular to it (figures 5A–E).

(ii) CPP crystals are positively birefringent, showing a lighter yellow colour when the long axis is
perpendicular to the compensator axis, and lighter blue than MSU crystals if parallel (figures 6A–
C).

(iii) All MSU crystals behave similarly in this system and being all similarly birefringent all clearly show
their negative birefringent sign. Not all CPP crystals clearly show their birefringent sign; many do
not show birefringence on simple polarised microscopy and may not show it with compensated
polarised microscopy. Other CPP crystals because of their shape are difficult to orient in relation
to the compensator axis, and occasional crystals may show negative birefringence.

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Figure 4: Simple polarised microscopy. (A, C) ×400; (B) ×600. Monosodium urate crystals are easily seen
because of ‘shine’ due to their strong birefringence. (A, B) Synovial fluid sample; (C) crystals obtained by
needling a tophus; these crystals are usually larger than those seen in synovial fluid. (For photography,
polarised filters are not totally crossed to allow some background detail.)

A B

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Figure 5: Compensated polarised microscopy. The arrow (λ) indicates the direction of the axis of the
compensator. (A, B) ×400 Synovial fluid: single monosodium urate (MSU) crystal oriented perpendicular and
parallel to the axis of the compensator. (C) ×400 Synovial fluid: multiple MSU crystals (and cells) showing
yellow or blue colour, depending on their orientation. (D, E) ×400 MSU crystals from needling two different
tophi. Size can be much larger than crystals found in synovial fluid. These figures show the appearance of
MSU crystals as seen by compensated polarised microscopy.

λ λ

A B

C D

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Figure 6: Compensated polarised microscopy ×1000. (A, B) Characteristic parallelepipedic calcium

pyrophosphate (CPP) crystals showing positive elongation (birefringence): blue if parallel and yellow if
perpendicular to the axis of the compensator (arrow (λ)). (C) Compensated polarised microscopy ×1000.
These abundant CPP crystals show the frequent difficulty of determining the sign of elongation of crystals
whose identity is clear from their shape. Crystals are here shown at ×1000 for educational purposes. Usually
observation is at ×400—or better at ×600—which allows good distinction of the crystals.

A B

The sample to be examined should be fresh, or at least examined during the day it is obtained, and if possible
kept at 4°C in a refrigerator to avoid decay of the cells, which is important, especially for the frequently
intracellular CPP crystals. For examination a small drop of SF fluid (a large drop makes the preparation too thick)
should be placed on a glass slide and a cover slip paced over it. No fixation or staining is required. The time spent
looking for crystals before deciding that a preparation is negative has not been critically ascertained; however,

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in most preparations crystals are abundant and often seen in the first field examined. Occasionally, fluids require
a prolonged examination.

If training with a compensated polarised microscope is started without first acquiring experience with an
ordinary microscope and then a simple polarised microscope this may cause problems, as a compensated
polarised microscope can be a difficult instrument to use.

Key references

Ivorra J, Rosas J, Pascual E. Most calcium pyrophosphate crystals appear as non-birefringent. Ann Rheum Dis
1999; 58 :582–4.

Lumbreras B, Pascual E, Frasquet J, et al. Analysis for crystals in synovial fluid: training of the analysts results in
high consistency. Ann Rheum Dis 2005; 64 :612–15.

Pascual E, Tovar J, Ruiz MT. The ordinary light microscope: an appropriate tool for provisional detection and
identification of crystals in synovial fluid. Ann Rheum Dis 1989; 48 :983–5.

Phelps P, Steele AD, McCarty DJ Jr. Compensated polarized light microscopy. Identification of crystals in
synovial fluids from gout and pseudogout. JAMA 1968; 203 :508–12.

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