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. Author manuscript; available in PMC: 2011 Jun 7.
Published in final edited form as: Bioanalysis. 2011 May;3(10):1085–1091. doi: 10.4155/bio.11.35

New approaches in the detection of calcium-containing microcrystals in synovial fluid

Aaron Hernandez-Santana 1, Alexander Yavorskyy 1, Sinéad T Loughran 2, Geraldine M McCarthy 3, Gillian P McMahon 1,†
PMCID: PMC3109550  EMSID: UKMS35452  PMID: 21585303

Abstract

Background

The presence of calcium phosphate crystals such as basic calcium phosphate and calcium pyrophosphate dihydrate in intra-articular fluid is linked to a number of destructive arthropathies and detection of these deposits is often pivotal for early diagnosis and appropriate management of such disease.

Results

We describe the use of a calcium-sensitive dye, Fluo-4, to selectively label calcium-containing mineral deposits in synovial fluid, which can then be easily visualized using a standard fluorescence microscope. Furthermore, we have combined the fluorescent properties of the tagged crystals with flow cytometry as a fast and semi-quantitative method of detection.

Conclusion

Dot-plots were used to quantify differences between various types of arthropathies and confirmed by visual observation of the crystals under a fluorescence microscope.

Arthritis is a painful medical condition involving damage to the joints in the body and a leading cause of disability. Synovial fluid (SF) analysis is one of the few clinical laboratory tests that is exclusively reserved for the diagnosis and assessment of the progression of arthritis [1]. In practice, only microbiological tests, white cell counts and examination for crystals are undertaken on a regular basis [2]. Microbiology tests are usually carried out to confirm and identify bacterial infection, and elevated white cell counts may also indicate inflammation or infection. The detection and identification of crystals such as monosodium urate (MSU) and calcium pyrophosphate dihydrate (CPPD) suggest the presence of acute gout and pseudogout (PG), respectively. Basic calcium phosphate (BCP), mainly composed of hydroxyapatite (HA), is another type of crystal found in up to 60% of SF samples from patients with osteoarthritis (OA) [3]. It is unclear whether BCP crystals are a cause or an effect in this condition, but it is established that their concentration increases with the severity of OA [4–6]. In this context, they appear to represent a potential therapeutic target for this disease. Other crystals, such as calcium oxalate (CO) can also occur in SF.

For the past half century, routine diagnosis of intra-articular crystal arthropathies has relied almost exclusively on the use of polarized light microscopy (PLM) to detect the presence of crystals from a sample aspirated from the affected joint [7]. Successful implementation of this technique in diagnosis requires adequate training as numerous reports have highlighted major flaws in this method, which can suffer from a large degree of bias and inconsistency [8–11]. For instance, only approximately one-fifth of all CPPD crystals actually show birefringence in practice, which questions the applicability of this technique [12]. Furthermore, amorphous BCP crystals are nonbirefringent and can be easily mistaken for debris [13].

Histological stains such as Alizarin Red and Von Kossa are sometimes used to help visualize calcium deposits in SF [14]. Calcium deposits appear either red (Alizarin) or deep grey/black (Von Kossa) when these stains are employed. These stains are still widely used, but their effectiveness is disputed since it often leads to discrepancies [7]. Despite efforts to develop new and reliable analytical techniques for studying intra-articular crystals, much work is still required [15–17].

In the area of bone research, calcein, xylenol orange, alizarin complexone and oxytetracycline have been used to label microdamage in bone, but these fluorochromes suffer from disadvantages such as limited aqueous solubility, high pKa value, low affinity for Ca2+ at neutral pH and high intrinsic fluorescence [18]. Further developments in this area led to the use of a set of fluorescent photoinduced electron transfer (PET) sensors such as Fluo-3 to selectively target scratches in bone [19]. Fluorescent calcium indicators were first described by Minta et al. in 1989, who synthesized a range of fluorescent dyes for the measurements of cytosolic free Ca2+ [20]. They combined the 8-coordinate tetracarboxylate chelating site of 1,2,-bis Z-aminophenoxyethane-N,N,N’,N’-tetraacetic acid (BAPTA) with xanthene chromophores to give rhodamine-like or fluorescein-like fluorophores. Over the years, a number of fluorescent dyes have been synthesized to suit different applications. Fluo-4 is an analogue of Fluo-3 with the two chlorine substituents replaced by fluorines (Figure 1) [21]. One of the crucial characteristics of this fluorochrome is that it undergoes a large fluorescent enhancement upon binding Ca2+, typically >100-fold, but with no accompanying spectral shift [22]. The excitation and emission wavelengths of Fluo-4 make it suitable for use in flow cytometry, fluorescence microscopy or microplate screening assays when used in conjunction with standard fluorescein filter sets. To this end, we have recently investigated the use of Fluo-4 to selectively stain calcium mineral deposits in SF.

Figure 1.

Figure 1

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Fluo-4 pentapotassium salt.

Experimental

Instrumentation & materials

Synthetic hydroxyapatite, CPPD, CO, MSU and HPLC-grade CHROMASOLV water were purchased from Sigma-Aldrich. Fluo-4 pentapotassium salt (F14200) was obtained from Invitrogen and stored in the dark at −21°C. Working solutions of the dye were thawed to room temperature and stored in the dark at 4°C thereafter. The potassium salt of this dye was used in order to provide water solubility.

Control SF samples from pigs were kindly supplied by Ann Rosenthal (Medical College of Wisconsin and the Zablocki VA Medical Center, Milwaukee, WI, USA). These SF samples were obtained from the hind legs of 300–500 lb adult pigs from a local slaughter house (Johnsonville Foods, Inc., Watertown, WI, USA). SF was removed from the knee joint with a 20 gauge needle and syringe and stored at 4°C. Patient SF samples were aspirated for therapeutic or diagnostic purposes by informed consent and were used in accordance with the ethical approval of the Mater Misericodiae University Hospital, Dublin, Ireland.

Phase contrast and fluorescence microscopy was carried out on a Leica DM1L inverted microscope. Fluorescence images were acquired using a standard fluorescein filter set. A FACSCalibur flow cytometer (BD Biosciences) was used in this study. The flowrate was set to 60 μl/min and forward scatter, side scatter and fluorescence data were collected over a period of 120 s. Fluorescence data was collected using the FL1 channel (with excitation provided by an air-cooled argon-ion laser (488 nm) and 530 nm emission band-pass filter (FITC). Data was processed using FACStation (BD Biosciences) software.

Fluo-4 staining of samples for fluorescence microscopy

Control SF (100 μl) was transferred to a clean eppendorf and mixed with a suspension of HA, CPPD, CO or MSU crystals in chromatography grade water (100 μl, 100 ppm). Fluo-4 (20 μl, 1 × 10−3 M) was added to the eppendorf tube and mixed at room temperature for 60 min. Samples were covered from light at all times. The sample was then centrifuged at 9000 rpm for 1 min and the supernatant removed. The pellet was resuspended in water (400 μl) and the centrifugation step repeated. The supernatant was removed, the pellet resuspended in water (100 μl) and a drop was then air-dried on a glass microscope slide for examination.

Fluo-4 staining of samples for flow cytometry

SF (100 μl, porcine or human) was placed in an eppendorf and diluted with water (400 μl). Fluo-4 (20 μl, 1 × 10−4 M) was added to the eppendorf tube and mixed at room temperature for 60 min. Samples were covered from light at all times. Samples were then transferred to plastic round bottom tubes (BD Falcon™) for use in the flow cytometer.

Results & discussion

Fluorescence microscopy of mineral deposits in synovial fluid

The composition of bone mineral is somewhat analogous to that of geological HA, the main difference being the substitution of other metals and carbonated groups in the crystal structure of most biological apatites [23]. Biologically derived HA surfaces are thought to contain Ca2+-rich surface layers at neutral pH [24]. Ca2+ ions are also thought to be present on the surface of CPPD and CO crystals, as revealed by studies of the mechanism of binding of phosphocitrate onto the surface of these minerals [25].

Parkesh et al. previously demonstrated that Fluo-3 and other similar dyes such as calcium crimson and calcium orange (based on different fluorophores) were capable of binding and ‘switching on’ upon binding to free Ca2+ at the site of microdamage in bone [19]. This ‘switching on’ or dramatic increase in fluorescence may be attributed to the disruption of the quenching effect of BAPTA upon binding to Ca2+ ions [26].

We decided to test the ability of Fluo-4 to bind to synthetic mineral crystals in SF, which is an extremely complex biological matrix. Porcine SF is well accepted in biomedical research as having very similar physiological composition of body fluids to humans [27]. Control SF was spiked with synthetic microcrystals of three calcium-containing minerals (HA, CPPD and CO) and MSU. MSU does not contain calcium, but is found in patients suffering from gout. Fluo-4 was added to the samples and incubated at room temperature with mixing to promote chemisorption of the dye on the crystal surface. A centrifugation and washing step was included to remove any unbound dye and minimize background fluorescence due to the binding of Fluo-4 to free Ca2+ ions in solution. Samples were covered in aluminum foil to avoid photodegradation of the fluorochrome. Samples of HA, CPPD and CO treated with Fluo-4 exhibited bright green fluorescence (Figure 2), most likely due to the binding of the dye to Ca2+ sites on the mineral surface. MSU showed no fluorescence as expected, which shows the selectivity of Fluo-4 towards calcium-containing minerals. No fluorescence was observed from the samples in the absence of the dye. Fluo-4 may also be used to stain microcrystals in human SF samples. In the example in Figure 3, the SF from a patient diagnosed with OA was stained with Fluo-4. Rectangular and rod-shape crystals were observed under phase-contrast microscopy (Figure 3A) and the corresponding fluorescence image (Figure 3B) showed bright green fluorescence emanating from these same structures, which suggests an interaction between the dye surface-bound Ca2+ ions.

Figure 2. Optical images of synovial fluid samples stained with Fluo-4 and spiked with (A) hydroxyapatite, (B) calcium pyrophosphate dihydrate, (C) calcium oxalate and (D) monosodium urate.

Figure 2

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Bright green fluorescence (A1–C1) was observed for all types of crystals except monosodium urate (D1), which lacks calcium.

Figure 3. Microscopic images of the synovial fluid from a patient diagnosed with osteoarthritis.

Figure 3

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(A) Phase contrast image and (B) corresponding fluorescence image.

Flow cytometry of synovial fluid

Flow cytometry is used routinely to rapidly analyze large numbers of cells individually using light-scattering, fluorescence and absorbance measurements [28]. One of the advantages of flow cytometry is that it can measure these parameters simultaneously, namely forward scatter, side scatter (90°) and fluorescence (typically four to six channels) per cell/particle, in contrast to bulk measurements. Measurements are made as the cells or particles flow through a small glass capillary in a fluid stream, and the fluidic system is designed in such a way that particles flow in ‘single file’. It can provide information on intrinsic structural parameters (i.e., cell size and shape, granularity, birefringence and photo synthetic pigments) as well as extrinsic functional and structural parameters (i.e., redox state, DNA and protein content and surface/intracellular antigens). ‘Forward scatter’ refers to the light scattered and detected in the forward direction, which correlates with particle size and refractive index. ‘Side scatter’, on the other hand, relates to the internal structure and granularity of the particle. Fluorescence measurements require the cell to be targeted with a fluorochrome label, which is usually delivered by means of an antibody, and are detected as the cells pass through the detector.

To date, flow cytometry of synovial fluid has been limited to the study of cells (i.e., lymphocytes [29], mesenchymal stem cells [30] and fibroblasts [31]) either derived from or present in the sample. Kaneko et al. used flow cytometry to detect small amounts of crystals produced in a supersaturated solution of uric acid with a view to understanding the crystallization mechanism in gouty arthritis, but these measurements were limited to in vitro studies [32].

While the staining of crystals described before helps identify calcium-containing mineral deposits in SF using simple fluorescence microscopy, there is still a need for fast, quantitative analytical techniques for the detection of microcrystals in SF. Following our microscopy studies, we envisaged that flow cytometry could deliver these requirements. First, the typical size of the crystals found in SF ranges from approximately 1 to 100 μm, similar dimensions to those of cells. Second, the excitation/emission profile of the Fluo-4 label on the crystals is compatible with the standard FITC optics of the flow cytometer, which allows us to collect fluorescence data.

To illustrate this principle, we added Fluo-4 to control SF and a series of patient SF samples diagnosed with either gout, rheumatoid arthritis (RA) or OA. Sample preparation was simple since no washing steps to remove unbound dye were required, as would be the case with non-PET dyes. Unlike the preparation of samples for microscopy, the SF sample is diluted by the sheath fluid as it is introduced into the optical detection chamber of the flow cytometer. Flow cytometry data are usually presented in histogram form or on a dot or contour plot. The frequency histogram is a direct graphical representation of the number of events occurring for each detection channel, while the dot plot is a 2D extension of the former. Each location on the dot plot above (Figure 4) corresponds to a measured signal at the forward scatter detector (y-axis) versus the fluorescence detector (x-axis).

Figure 4. Representative dot-plots of control and patient synovial fluid samples after flow cytometry, and their corresponding fluorescence images.

Figure 4

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(A) Control synovial fluid, (B) gout, (C) rheumatoid arthritis and (D) osteoarthritis/pseudogout.

Representative dot-plots and fluorescence images of four different samples are presented in Figure 4. Control SF showed a relatively large number of events with a high forward scatter but with low fluorescent character, which suggests that the particles were practically non-fluorescent at the measured wavelength. The composition of these particles is unknown but fluorescence microscopy images of the same sample were consistent with the lack of calcium-containing particles (Figure 4A). Gout and RA are regarded as inflammatory arthropathies and should therefore rarely contain calcium deposits in their fluids [33], as confirmed experimentally (Figure 4B & C). On the other hand, OA is commonly associated with the presence of calcium phosphate deposits. OA samples run in this study typically showed a large number of events with a large fluorescent component, as expected (Figure 4D).

The relative number of total events and fluorescent events are described in Table 1. OA (n = 11) and RA (n = 3) samples showed the highest total number of events, but these were very low in gout (n = 3). Our real interest was in the relative number of fluorescent events, since these carry the Fluo-4 tag, which provides an additional degree of discrimination through its binding to calcium-containing crystals. By setting a reference threshold on the fluorescence channel (x-axis) based on the control SF control, we can compare the relative number of fluorescence events for each sample. OA SF samples showed approximately ten- and 100-times the average number of fluorescent events compared with RA and gout samples, respectively, which suggests a comparatively large number of calcium-containing particulates in the sample.

Table 1.

Flow cytometry results for control SF and patient SF.

Description Number of
samples		 Total number
of events† 		Number of fluorescence
events‡
Control SF 1 1335 1
Gout 3 100 ± 41 15 ± 5
RA 3 6000 ± 5550 87 ± 43
Osteoarthritis/PG 11 8362 ± 3561 1085 ± 471
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†

Runtime was 120 s.

‡

Threshold was set at a channel number of 90 counts in the fluorescence channel.

PG: Pseudogout; RA: Rheumatoid arthritis; SF: Synovial fluid.

Conclusion

Fluo-4, a calcium-sensitive dye, was used to stain calcium-containing mineral deposits in SF. The staining protocol is simple and is carried out at room temperature and physiological pH. Unlike traditional SF staining methods, the dye is only ‘switched on’ when bound to calcium-containing minerals, which provides an extra degree of chemical specificity and results in easier discrimination.

To our knowledge, this is the first time that flow cytometry has been used explicitly to detect calcium-containing crystals in SF. Fluorescently tagged microcrystals were detected using a standard fluorescence channel in the flow cytometer, providing quantitative information on the relative number of calcium-containing particles in the sample. Using dot-plots, we were able to detect large differences between different arthropathies (gout, RA and OA), and high fluorescence counts correlated with the diagnosis and visual observation of crystals under the microscope. We envisage that further development of this technique and the study of a larger pool of patient SF samples could lead to a quick, quantitative analytical technique to screen different forms of arthritis, ultimately helping support clinical diagnosis and the selection of an appropriate treatment.

Future perspective

The lack of appropriate analytical techniques to analyze SF and support other clinical observations may often lead to misdiagnosis and inappropriate treatment. There is a clinical need for quick and simple diagnostic tests for joint fluid crystal identification. Ultimately, advances in this area need to be forged through collaborations between clinicians and basic scientists. Flow cytometry is routinely used in many clinical and research laboratories, and we envisage that further development and validation of this technique could lead to a cost effective and rapid screening test for microcrystals in SF.

Executive summary.

  • ▪ Detection of intra-articular crystals is a useful diagnostic parameter in the clinical diagnosis and management of a range of arthropathies.

  • ▪ Fluo-4, a calcium-sensitive dye, can be used to selectively label calcium-containing mineral deposits in synovial fluid (SF).

  • ▪ Fluorescence microscopy of the stained crystals requires minimal training and can provide quick and simple visualization of mineral deposits in a SF sample.

  • ▪ Flow cytometry, a routinely used fluorescence-based technique, may be used to obtain quantitative data with regard to the relative number of mineral deposits in a SF sample.

  • ▪ Using dot-plots, we were able to detect large differences between different arthropathies (gout, rheumatoid arthritis and osteoarthritis), and high fluorescence counts correlated with the diagnosis and visual observation of crystals under the microscope.

  • ▪ Osteoarthritis SF samples, in particular, showed approximately ten and 100-times the average number of fluorescent events compared with rheumatoid arthritis and gout samples, respectively, which suggests a comparatively large number of calcium-containing particulates in the sample.

Acknowledgments

The authors would like to thank Finbarr O’Sullivan (NICB) for help and access to the fluorescence microscope.

The authors would like to acknowledge the financial support from the Wellcome Trust (Grant No. WT081091).

Key Terms

Arthritis

Group of painful conditions involving damage to the joints of the body.

Synovial fluid

Viscous fluid secreted by the synovial membrane typically found between the joint cavities that serves to lubricate the joints and provide nutrients to the cartilage.

Calcium

Most abundant mineral in the human body, mainly found in bones and teeth.

Fluorescence

Emission of light by a substance following absorption of electromagnetic radiation of a different wavelength.

Flow cytometry

Powerful analytical technique for counting and examining the properties of individual cells or particles.

Microcrystals

Microscopic crystals that are normally visualized with the aid of a microscope.

Footnotes

Ethical conduct of research The authors state that they have obtained appropriate institutional review board approval or have followed the principles outlined in the Declaration of Helsinki for all human or animal experimental investigations. In addition, for investi gations involving human subjects, informed consent has been obtained from the participants involved.

Financial & competing interests disclosure Sinéad Loughran would like to acknowledge funding from the Health Research Board, Ireland. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

No writing assistance was utilized in the production of this manuscript.

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