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How to perform and analyse synovial biopsies
Best Practice & Research Clinical Rheumatology 27 (2013) 195–207
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How to perform and analyse synovial biopsies Daniëlle M. Gerlag, MD PhD a, *, Paul P. Tak, MD PhD a, b, c a
Division of Clinical Immunology and Rheumatology, Academic Medical Centre/University of Amsterdam, F4-105, P.O. Box 22700, 1100 DE Amsterdam, The Netherlands b University of Cambridge, Cambridge, UK c GlaxoSmithKline, Stevenage, UK
a b s t r a c t Keywords: Arthritis Synovitis Synovial tissue Synovial biopsy Arthroscopy Synovial biomarkers Digital image analysis
Although most of the rheumatologic diseases can be diagnosed based on clinical examination combined with additional laboratory and radiographic tests, histological examination of synovial tissue may lead to the correct diagnosis and adjustment of therapy when neoplastic or granulomatous disease, deposition disease or infection in spite of negative synovial fluid culture is suspected. For research purposes synovial tissue analysis is used to investigate the pathological changes of the synovium in studies aimed at elucidating the aetiology and pathogenetic mechanisms involved in arthritis. In addition, the use of synovial biomarkers has been shown to be instrumental in the developmental process of new therapeutics. In this chapter, several minimally invasive techniques for acquiring synovial tissue samples, handling of the tissue and the analysis thereof are described. Ó 2013 Elsevier Ltd. All rights reserved.
Synovial biopsies for clinical practice For most rheumatologic diseases, the diagnosis can be made on the basis of clinical examination, routine laboratory tests, radiographic examination and the analysis of synovial fluid if present. Examination of the aspirated fluid from the joint cavity can help to make a distinction between inflammatory and non-inflammatory arthropathies, and when a crystal-induced or bacterial arthritis is suspected, analysis of the synovial fluid is essential in establishing the diagnosis. When synovial fluid cannot be aspirated, however, or in case of unclassified arthritis, suspicion of neoplastic or granulomatous disease, deposition disease or infection in spite of negative synovial fluid * Corresponding author. Tel.: þ31 20 5667765; fax: þ31 20 6919658. E-mail addresses: [email protected] (D.M. Gerlag), [email protected] (P.P. Tak). 1521-6942/$ – see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.berh.2013.03.006
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culture, then histological examination of synovial tissue may lead to the correct diagnosis and adjustment of therapy [1]. Analysis of synovial biopsy samples can assist in the detection of joint infections. In acute bacterial infections, the tissue usually contains many polymorphonuclear leucocytes, and Gram’s staining sometimes reveals bacteria. When blood and synovial fluid cultures are negative, culture of tissue biopsies may still be positive. Using broad-range bacterial primers to analyse and amplify gene coding for ribosomal RNA (16S rRNA) by polymerase chain reaction (PCR) has been successful in demonstrating bacterial species when cultures were negative [2]. Especially when antibiotic treatment has been initiated before culturing synovial fluid and in case of micro-bacterial infections with bacteria that are difficult to culture or grow slowly, this technique can help in establishing the correct diagnosis [3]. Specific infections, such as mycobacterial infections, can lead to specific histological changes in the synovium. Granulomas and acid-fast organisms may be found in these cases. Applying mycobacterial genus-specific PCR on directly isolated DNA extracts from the tissue may help to demonstrate the infectious agent [4]. Appropriate staining may also allow the detection of other microorganisms, such as fungi and spirochaetes (Lyme disease and syphilis). Gout and pseudo-gout can lead to deposits of tophus-like material in the synovial membrane and cartilage. When synovial fluid analysis is repeatedly negative, tissue examination may be of help in diagnosing these deposition diseases. When gout is suspected, the tissue should be conserved in absolute alcohol because the monosodium urate crystals will dissolve in most other fixatives. Unstained sections can be examined with the polarisation microscope, but using DeGolanthal stain for urate is also possible. In amyloidosis, specific amyloid deposits can be found in the synovial tissue. These deposits will colour pink with haematoxylin–eosin and red with Congo red staining and can be surrounded by macrophages, multinucleated giant cells and granulation tissue. Other pigments can be found if appropriate staining is applied, such as in the case of ochronosis, haemochromatosis as well as in recurrent haemarthrosis. In synovial chondromatosis, pigmented villonodular synovitis (PVNS) and multicentric reticulohistiocytosis specific changes of the synovial tissue histology can be found. Other specific changes can be found in the case of synovial chondrosarcomas, synovial haemangiomas, lipoma arborescens and intracapsular chondromas, as well as in Erdheim–Chester disease, sarcoidosis and arthritis caused by foreign-body material [5]. Taken together, examination of synovial tissue may help to make a diagnosis in selected cases of infectious, infiltrative and deposition diseases of joints.
Practice points Synovial tissue analysis may assist in the diagnostic work-up in the case of suspicion of neoplastic or granulomatous disease, deposition disease or infection in spite of negative synovial fluid culture. Synovial biopsies for research purposes Examination of the synovial tissue is also used to investigate the pathological changes of the synovium in studies aimed at elucidating the aetiology and pathogenetic mechanisms involved in arthritis. Descriptive studies of the cell infiltrate in the synovial tissue have provided insight into the pathogenesis and contributed greatly to the understanding of the role of different cell types and various mediators in various forms of arthritis. As a result, this knowledge has led to the identification of potential new targets of therapy for these diseases. Differentiation of early arthritis Although analysis of the synovial tissue could conceivably be helpful in establishing an early diagnosis in patients presenting with immune-mediated arthritis of recent onset, many of the pathological changes found in the inflamed synovium are not specific for any form of arthritis.
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Nonetheless, synovial tissue analysis might have diagnostic potential in distinguishing for instance rheumatoid arthritis (RA) from other forms of arthritis, which would be important in patients presenting with mono- or oligoarthritis, before they fulfil the classification criteria for RA. This is of importance knowing the evidence that early therapeutic intervention with disease-modifying antirheumatic therapies delays or inhibits structural damage in this disease. Being able to use the so-called ‘window of opportunity’ in these patients, while not overtreating other patients who will not develop RA, would be a major step forward towards personalised medicine. From early arthritis clinics it is known that in about 30% of the patients the diagnosis of RA can be established using the 1987 American College of Rheumatology (ACR) criteria at the time of presentation. Another 30% of the patients have an unclassified arthritis [6]. Of these patients, some will develop RA during the follow-up period. Several factors, such as the presence of various autoantibodies, can help to identify the patients who will develop RA using the recently published 2010 American College of Rheumatology/European League Against Rheumatism (ACR/EULAR) criteria, meant to diagnose RA earlier [7,8]. In the synovial tissue, massive infiltration by plasma cells and macrophages in the synovial sublining can predict the diagnosis of RA solely on the basis of examination of synovial biopsy specimens with an accuracy of 85% (Fig. 1). A diagnosis other than RA can be predicted in 97% of the cases when minimal infiltration by these cells is found. These variables were identified using multivariate models in a study of 95 patients with early unclassified arthritis, defined by a disease duration of <12 months [9]. In another study of 71 patients, the intensity of B- and T-cell infiltration and the expression of the av integrin could be used to differentiate patients with RA from those with spondyloarthritis and osteoarthritis [10] (Fig. 2). Other reports have suggested that lining layer depth and the number of CD163positive macrophages might help to distinguish between juvenile-onset spondyloarthritis and other juvenile idiopathic arthritis subsets [11] (Fig. 3). Furthermore, enhanced expression and activation of various protein kinases and factors involved in angiogenesis in the synovial tissue of patients with unclassified arthritis can predict the development of RA and erosive disease in these patients [12]. Taken together, there is some evidence that synovial tissue analysis could help to distinguish between different forms of immune-mediated inflammatory arthritis, but at this moment none of the available tests would justify synovial biopsy to establish the diagnosis of RA or spondyloarthritis in clinical practice. The challenge for the near future will be to explore the value of more sophisticated tools for synovial tissue analysis, such as microarray analysis and proteomics. This approach could provide insight into the pathogenesis of different arthritides as well as distinct subsets of RA leading to common signs and
Fig. 1. CD38 positive plasma cells in the inflamed synovial tissue of an RA patient (100 ).
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Fig. 2. CD 3 positive lymphocytes in the synovial tissue of a patient with active rheumatoid arthritis (100 ).
symptoms [13] and may also lead to the identification of novel diagnostic and prognostic markers that may be used to identify patients who are at risk of having persistent and destructive disease. In the context of personalised medicine, such tools could guide the initiation of tailor-made targeted therapies to prevent autonomous disease progression and irreversible joint damage by early intervention.
The use of synovial biomarkers in the evaluation of novel therapies Synovial biomarkers have been used for various goals, including differential diagnosis, prediction of radiological outcome, evaluation of novel therapies and prediction of clinical response to treatment. At
Fig. 3. CD 55 positive fibroblast like synoviocytes in the inflamed RA synovial tissue (100 ).
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Fig. 4. Minimally invasive needle-arthroscopy of the left knee. Biopsies are taken through an infra-patellar portal and kept on a wet gauze.
present synovial biomarkers are mainly used for analysis on the group level. Conceivably, synovial biomarkers might also be developed in the future to use as surrogate markers to predict outcome in individual patients. Recent work has shown that the use of synovial biomarkers is instrumental in the developmental process of new therapeutics. An advantage is that an early therapeutic effect in the target tissue, the synovial tissue, can be detected in relatively small numbers of patients. Therefore, this approach can be used to screen for potential therapeutic effects, which may accelerate decisions in phase I/II clinical trials. In addition, dose selection may be enhanced before large, conventional clinical trials are conducted. If there is initial proof of concept, such high density of data studies provides the rationale for phase III trials that are necessary to determine whether the biological effects found in these earlier studies translate into clinically meaningful improvement. Furthermore, the description of changes after specific interventions provides insight into the mechanism of action of the treatment, as well as into the role of specific cells and molecules in the pathogenesis of the disease studied, leading to discovery of potential targets for novel therapies. The need for the use of biomarkers in this context is becoming increasingly clear from the large number of compounds in the pipeline of pharmaceutical industry, the increasing difficulty including patients with active arthritis in clinical trials due to the success of available treatment as well as financial and ethical reasons [14]. Early proof-of-concept studies including serial synovial biopsies obviously provide data on the specific mechanism of action of a specific intervention. An example would be the evaluation of B cells in different compartments including the synovium after rituximab treatment [15]. In addition, biomarkers that respond to treatment independent of the specific mechanism of action may be measured. Recent work has shown that it is indeed possible to use such biomarkers [16–18], that serve as a sensitive tool when used for selection purposes in early clinical trials [14]. In RA most of the data are available for the number of the CD68-positive macrophages in the synovial sublining of patients with RA, which has been demonstrated to discriminate between effective treatment on the one hand versus ineffective and placebo treatment on the other [17,18]. It should be noted that it is critical to use standardised and validated techniques to detect and quantify the number of CD68-positive macrophages to get reliable results, but this is clearly feasible. Similarly, it has recently been suggested that the number of CD3-positive T cells distinguishes between effective treatment and placebo treatment in patients with psoriatic arthritis, although the data are still limited [19–21].
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Practice points Synovial tissue analysis could help to distinguish between different forms of immunemediated inflammatory arthritis, but at this moment none of the available tests would justify synovial biopsy to establish the diagnosis in clinical practice. Evaluation of serial synovial biopsies may assist in screening for potential efficacy of novel compounds and provide insight into the mechanism of therapy.
Techniques to obtain synovial tissue Various minimally invasive techniques are being used to access synovial tissue, of which the blind needle and arthroscopic techniques are most common. More recently, ultrasound-guided biopsy techniques have been introduced. All these techniques are generally well tolerated and can be performed safely in an outpatient setting. Blind needle biopsy One of the methods to acquire synovial tissue being described in earlier studies is the blind or closed needle biopsy technique, using the Parker-Pearson needle under local anaesthesia. This needle is a simplified 14-gauge biopsy needle that does not require a skin incision. It is a safe, well-tolerated, technically easy and inexpensive method that can be performed in the outpatient setting. After cleansing, the skin, subcutaneous tissue and joint capsule are anaesthetised using 1–2% lidocaine. Following adequate anaesthesia, the trochar is inserted into the joint and the biopsy needle is introduced into the trochar. Multiple biopsies from different locations throughout the joint can be obtained by changing the angle of the needle. When suction is applied to a Luer-Lock syringe attached to the biopsy needle, the tissue can be obtained more easily and filling the joint cavity with 10–20 ml isotonic saline before introducing the trochar may facilitate the procedure in the case of minimal or no clinical evidence of effusion. This technique is mostly used to biopsy larger joints, such as the knee joint, but its application to other joints, including the ankle, wrist, elbow and shoulder, have also been described. A modified shorter needle can be used to access smaller joints. No complications, such as haemarthrosis, have been reported in a series of >800 samples [22]. Modifications to this technique have been described, for instance, using the Tru-Cut needle. This is an inexpensive, disposable item used for a variety of soft-tissue biopsies that can also be used to retrieve synovial tissue through a procedure similar to that described above. In most cases, this technique yields adequate tissue samples, but it can fail when joints are not swollen. Furthermore, it is usually restricted to larger joints, such as the knee joint, and potential sampling error can be introduced because the operator is not able to visually select the tissue. Another disadvantage of the blind needle biopsy technique is that it is not always possible to obtain adequate tissue samples, especially in the case of biopsy of clinically quiescent joints, for example, after successful therapy. Arthroscopic biopsy Another method to obtain synovial tissue that made its entrance in rheumatology during the 1970s is the use of high-definition, small-bore arthroscopy. Various-sized arthroscopes can be used for different joints. Knee arthroscopy can be performed when a small-bore arthroscope or needlearthroscope, with a diameter ranging from 4.5 to 2.7 mm, is introduced into an infrapatellar skin portal, created after disinfecting the skin and anaesthetising the skin, subcutis and knee joint. For the grasping forceps, a suprapatellar second skin portal is installed. This technique can also be applied
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when biopsy samples of an ankle joint are taken, using a medial and lateral malleolar portal. A small-bore arthroscope of 1.9 mm can be used in the case of even a smaller joint, such as the metacarpophalangeal joint or the wrist, through a radial and ulnar skin portal [23]. Since the introduction of local and regional anaesthetics, this procedure can be performed safely in an office-based setting [24] (Fig. 4). There are several potential advantages of this technique when compared to the blind needle biopsy procedure. One of these advantages is the ability to evaluate the synovium and cartilage macroscopically. Studies have shown that macroscopic signs of inflammation at the biopsy site are positively correlated to the immunohistological features of inflammatory activity in the corresponding tissue sample, although the correlations are only very moderate [25,26]. It is important to realise that it is not possible to predict in a reliable way the microscopic features of the inflamed synovium on the basis of the macroscopic appearance at arthroscopy. Although previous reports suggested that macroscopic scoring systems could be used to distinguish different patient groups, these visual scoring systems still need to be validated. In addition, the evaluation of the articular cartilage by arthroscopy could potentially be used for the evaluation of treatment effects, but this method also requires further standardisation and validation. As mentioned above, another advantage of arthroscopy over blind needle biopsy is the possibility to evaluate smaller joints, such as ankles, wrists and even metacarpophalangeal and proximal interphalangeal joints, using arthroscopes with a narrow diameter. This allows the analysis of synovium in an early stage of disease and in the case of a polyarthritis of smaller joints. The measures of inflammation in tissue samples taken from clinically inflamed joints are generally similar irrespective of being obtained through the blind needle technique or by arthroscopy under visual inspection [27]. However, an important difference between the two methods is that it is always possible to obtain adequate amounts of synovial tissue by arthroscopy, even when the volume of the tissue has decreased following effective treatment. The arthroscopic procedure performed by an increasing number of rheumatologists in an outpatient setting under local or regional anaesthesia is well tolerated by the patients and has a low complication rate. Minimal pain or discomfort during the procedure was reported by 35–36% of the patients in a survey among rheumatologists performing this procedure [28]. Minor complications, such as vasovagal reactions and temporary swelling of the joint, were mentioned in 5–10% of the cases. In a recent survey, in which information of 15,682 arthroscopies performed by rheumatologists was collected, the complication rate of haemarthrosis was 0.9%, deep vein thrombosis 0.2% and wound and joint infection 0.1% [24]. The total number of complications reported was 15.1 per 1000 arthroscopies, which is comparable to the figures reported in the orthopaedic literature. The irrigation volume of the knee joint was positively correlated with the rate of wound infection and the total complication rate, possibly as a result of the extended length of the procedure. However, joint lavage may also have positive effects. Arthroscopic joint lavage In the case of persisting arthritis after needle aspiration and the administration of local corticosteroids, joint lavage through arthroscopy can be considered. Removal of pro-inflammatory and destructive mediators as well as cartilage degradation products is the probable reason for the therapeutic effect seen after this procedure. It should be noted, however, that the effect might differ between different forms of arthritis. Although various uncontrolled, retrospective studies have suggested a beneficial therapeutic effect of joint lavage for osteoarthritis of the knee joint, a randomised, placebocontrolled trial did not show superiority of this procedure compared to placebo [29]. For inflammatory arthritis, such as RA, the efficacy of joint lavage appears more promising. Retrospective analysis of the therapeutic effect of needle aspiration or joint lavage in 50 patients did reveal a superior effect of arthroscopic lavage [30]. This effect seems to be dependent on the volume of physiological fluid used during the procedure, with 5–10 l giving the best results [31,32]. Of importance from the methodological point of view, lavage should be avoided when serial biopsies are taken to evaluate the effects of specific treatments, as the results may be biased due to the anti-inflammatory effect of the lavage procedure.
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Ultrasound-guided synovial biopsy Synovial biopsies will probably increasingly be obtained by means of more recently developed office-based mini-invasive, ultrasound-guided techniques [33]. During this procedure, synovial tissue is acquired percutaneously using a portal and a rigid forceps technique [34]. This approach combines the advantages of being minimally invasive and being able to sample inflamed synovial tissue under indirect visual inspection. Using a multifrequency linear transducer, effusion and synovitis can be identified. The sensitivity can be enhanced applying power-Doppler techniques [35]. Under standard sterile conditions and after infiltrating the skin and subcutaneous tissue with local anaesthetics, a 14gauge needle can be inserted into the designated area under ultrasound guidance. After creating a portal, by means of a flexible wire, followed by a percutaneous sheath, synovial samples can be acquired using a rigid forceps [33,34]. The association of ultrasound-determined pathological synovial blood flow and synovial pathology has been established [36].
Practice points Needle biopsy is simple and cheap. Arthroscopic biopsy is technically more complicated but provides adequate synovial tissue samples, even from clinically uninvolved joints, selected under direct vision from both large and small joints. Synovial biopsies will probably increasingly be obtained by means of more recently developed office-based mini-invasive, ultrasound-guided techniques.
Handling of the tissue Depending on the reason for the biopsy procedure, being diagnostic or for research purposes, the tissue needs to be handled differently. For routine pathological examination by light microscopy, the tissue samples should be fixed in 4% formalin and embedded in paraffin. This can be used for haematoxylin and eosin staining and certain immunohistochemical stainings. When gout is suspected, the synovial tissue should be conserved in absolute alcohol because the monosodium urate crystals will dissolve in most other fixatives. Unstained sections can be examined by polarisation microscopy, but using DeGolanthal stain for urate is also possible. In the case of a suspected infection, the tissue should be kept in suitable culture media. Progress has also been made in the use of PCR on synovial biopsies for detection of bacterial DNA [37]. Here, contaminating DNA needs be avoided and the samples should be snap frozen in liquid nitrogen. For research purposes synovial tissue can be processed for histology, immunohistochemical staining, immunofluorescence, in situ hybridisation, PCR, micro-array, proteomics and cell or tissue culture. Immunohistochemistry can be performed on formalin-fixed, paraffin-embedded material or on samples that were snap frozen in optimal cutting temperature (OCT) embedding medium and stored in liquid nitrogen. Detection of messenger RNA (mRNA) is possible in samples that are snap frozen immediately after the tissue is obtained. It is also possible to culture synovial cell populations and whole biopsy samples in tissue culture media [38]. Tissue heterogeneity and representativeness of biopsy samples Most of the data on this subject have been retrieved from synovial tissue of RA patients. Several studies have suggested a degree of morphological heterogeneity in synovial tissue samples taken from one joint [25]. It is possible, however, to quantify several markers of inflammation in a reliable way by examining only a limited area of tissue [39–41]. For T-cell infiltration and expression of activation antigens in RA synovium, a variance of <10% can be reached when at least six biopsy specimens are examined [42], suggesting that representative data can be obtained when a limited number of biopsy
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samples from different areas within one joint are investigated. When the biopsies are taken form an actively inflamed joint, there is on average no difference in the features of synovial inflammation at the pannus–cartilage junction (where the inflamed synovial tissue invades into the cartilage and underlying bone) compared with other regions of the joint [43,44]. A recent paper described the immunohistochemical assessment of the synovial tissue obtained by ultrasound-guided biopsy from the small joints of nine patients with RA. This study showed that acceptable variance was reached when at least eight tissue samples were obtained from one small hand joint to minimise sampling error [33]. The signs of inflammation in tissue samples taken from inflamed small joints compared to larger joints, such as the knee joint, are generally comparable [23], indicating that the inflammation in one inflamed joint is representative of that in other inflamed joints. By contrast, there is large variability of synovial inflammation between different patients in all phases of the disease [45–48]. It is therefore critical that the number of patients is sufficient when different conditions are being compared. In addition, patient cohorts should be stratified for use of medication and disease activity to allow meaningful conclusions based on the analysis of synovial biopsies.
Practice points Depending on the indication for the biopsy procedure the tissue needs to be handled differently. In order to minimise sampling error, at least six to eight biopsy samples from different areas throughout the joint need to be taken. Stratifying for disease activity and use of medication is essential when the features of synovial inflammation are compared between different patient groups. In RA the inflammation in one inflamed joint is generally comparable to that in other inflamed joints. Analysis of the synovial tissue Depending on the purpose of the analysis of the synovial tissue, different methods can be used. Some of these are briefly discussed in this review article [49]. Quantification of inflammation by histology and immunohistochemistry For quantification of synovial inflammation, three methods have been used. The first method, conventional quantitative analysis by counting cells, is reliable but time consuming. In the evaluation of large series of tissue sections, this is an important disadvantage. As it can be anticipated that the analysis of synovial tissue samples will be increasingly incorporated into clinical trial design, other, less time-consuming methods, need to be applied. A method that is more time efficient is semiquantitative analysis. For instance a 5-point scale (ranging from 0 to 4) by several observers can be used and minor differences between observers can be resolved by mutual agreement [50]. Alternatively, the average score could be calculated. This method has been used in many studies [27,45] and is less sensitive to detect small changes than fully quantitative analysis (by definition) [51]. A third technique, digital image analysis, combines the advantages of these two methods, being both sensitive and time efficient [50,52,53]. We use this computer-assisted approach to evaluate 18 randomly selected high-power fields. By marking the intimal lining layer of the synovial tissue, separate analysis of the intimal lining layer versus the synovial sublining can be performed. In this way, positively stained cells as well as the optical density of the stained area can be measured per square millimetre. This technique has been shown to be reliable for the analysis of the cell infiltrate and the expression of cytokines, matrix metalloproteinases, vascular markers, adhesion molecules and chemokines [41,50]. Inter- and intrarater reliability using intraclass correlations showed good reliability and the measurement of change in total positive cell numbers in synovial tissue can be determined reproducibly for various cell types in RA clinical trials [54].
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Gene expression profiling For gene expression analysis on synovial biopsies, a validated real-time quantitative PCR (Q-PCR) on small synovial tissue samples can be used. This method was developed to complement immunohistochemical analysis and uses a cell-based standard curve, generated with complementary DNA (cDNA) derived from activated peripheral blood mononuclear cells. This approach significantly improved the assay reliability by reducing variation and by simplifying assay development. When this method is applied, six or more synovial tissue samples should be pooled to limit sampling error [55]. A method to analyse not only a selected set of genes but also potentially all genes expressed in the synovial tissue is microarray analysis. Using gene expression profiles determined with cDNA microarray analysis, characterisation of specific signatures can lead us to unravel heterogeneous diseases such as RA and spondyloarthritis. Analysis of the expression profiles with a focus on immune-related genes in synovial tissues from patients with RA revealed considerable variability, resulting in the identification of various molecularly distinct forms of this disease [13,56]. It can be anticipated that studies using this approach will deepen the insight into the molecular mechanisms underlying different pathogenetic subsets of RA and might also facilitate stratification in clinical trials. In addition to gene expression profiling of whole tissue samples, it is also possible to examine specific areas selected in tissue sections by laser-mediated microdissection (LMM) [57]. Target areas of cryosections are cut out using a combination of a microscope and a laser-beam generator. Using a nonfocussed laser beam without direct contact to avoid contamination (a process called laser pressure catapulting), tissue compartments or single viable cells can be isolated. This technique has already been used and validated in several studies in rheumatology [58,59]. Combined with the techniques described above, LMM may be a very interesting and novel way to analyse gene expression in tissues form patients with rheumatological diseases.
Practice points For quantification of synovial inflammation digital image analysis is sensitive and time efficient. Quantitative PCR and various microarray analysis techniques can be used to analyse synovial tissue samples for research purposes.
Conclusion Synovial tissue analysis can be of help in establishing a rheumatological diagnosis and may lead to adjustment of treatment in selected cases in clinical practice. Synovial tissue analysis is especially used for research purposes. It is easily accessible and can be acquired using various minimally invasive techniques in a safe office-based setting. The handling and analysis of the tissue depend on the specific question that needs to be answered. In all cases, standardisation and validation of techniques are critical, but previous work has clearly shown that reliable synovial tissue analysis is feasible and may reveal a wealth of information.
Research agenda develop and determine the value of new tools for synovial tissue analysis; identify novel diagnostic and prognostic markers in the synovial tissue; and analyse synovial tissue in individuals at risk of RA and very early RA.
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Selective involvement of ERK and JNK mitogen-activated protein kinases in early rheumatoid arthritis (1987 ACR criteria compared to 2010 ACR/EULAR criteria): a prospective study aimed at identification of diagnostic and prognostic biomarkers as well as therapeutic targets. Annals of the Rheumatic Diseases 2012;71(3):415–23. [13] van der Pouw Kraan TC, van Gaalen FA, Kasperkovitz PV, Verbeet NL, Smeets TJ, Kraan MC, et al. Rheumatoid arthritis is a heterogeneous disease: evidence for differences in the activation of the STAT-1 pathway between rheumatoid tissues. Arthritis and Rheumatism 2003;48(8):2132–45. *[14] Gerlag DM, Tak PP. Novel approaches for the treatment of rheumatoid arthritis: lessons from the evaluation of synovial biomarkers in clinical trials. Best Practice and Research Clinical Rheumatology 2008;22(2):311–23. [15] Thurlings RM, Vos K, Wijbrandts CA, Zwinderman A, Gerlag DM, Tak PP. Synovial tissue response to rituximab: mechanism of action and identification of biomarkers of response. Annals of the Rheumatic Diseases 2008;67(7):917–25. [16] Gerlag DM, Haringman JJ, Smeets TJ, Zwinderman AH, Kraan MC, Laud PJ, et al. Effects of oral prednisolone on biomarkers in synovial tissue and clinical improvement in rheumatoid arthritis. Arthritis and Rheumatism 2004;50(12): 3783–91. [17] Haringman JJ, Gerlag DM, Zwinderman AH, Smeets TJ, Kraan MC, Baeten D, et al. Synovial tissue macrophages: a sensitive biomarker for response to treatment in patients with rheumatoid arthritis. Annals of the Rheumatic Diseases 2005;64(6): 834–8. *[18] Wijbrandts CA, Vergunst CE, Haringman JJ, Gerlag DM, Smeets TJ, Tak PP. Absence of changes in the number of synovial sublining macrophages after ineffective treatment for rheumatoid arthritis: Implications for use of synovial sublining macrophages as a biomarker. Arthritis and Rheumatism 2007;56(11):3869–71. [19] van Kuijk AW, Gerlag DM, Vos K, Wolbink G, de GM, De Rie MA, et al. A prospective, randomized, placebo-controlled study to identify biomarkers associated with active treatment in psoriatic arthritis: effects of adalimumab treatment on synovial tissue. Annals of the Rheumatic Diseases 2008;68(8):1303–9. [20] Pontifex EK, Gogarty M, Gibbs A, Fearon U, Veale DJ, Bresnihan B, et al. Change in CD3 positive cell infiltration in psoriatic arthritis (PsA) synovial tissue correlates with change in DAS28 following initiation of biologis therapy. Annals of the Rheumatic Diseases 2008;67(Suppl. II):628. [Ref Type: Abstract]. [21] Fitzgerald O, Chandran V. Update on biomarkers in psoriatic arthritis: a report from the GRAPPA 2010 annual meeting. Journal of Rheumatology 2012;39(2):427–30. [22] Gerlag D, Tak PP. Synovial biopsy. Best Practice and Research Clinical Rheumatology 2005;19(3):387–400. *[23] Kraan MC, Reece RJ, Smeets TJ, Veale DJ, Emery P, Tak PP. Comparison of synovial tissues from the knee joints and the small joints of rheumatoid arthritis patients: implications for pathogenesis and evaluation of treatment. Arthritis and Rheumatism 2002;46(8):2034–8. [24] Kane D, Veale DJ, Fitzgerald O, Reece R. Survey of arthroscopy performed by rheumatologists. Rheumatology (Oxford) 2002;41(2):210–5.
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[25] Lindblad S, Hedfors E. Intraarticular variation in synovitis. Local macroscopic and microscopic signs of inflammatory activity are significantly correlated. Arthritis and Rheumatism 1985;28(9):977–86. [26] Rooney M, Condell D, Quinlan W, Daly L, Whelan A, Feighery C, et al. Analysis of the histologic variation of synovitis in rheumatoid arthritis. Arthritis and Rheumatism 1988;31(8):956–63. *[27] Youssef PP, Kraan M, Breedveld F, Bresnihan B, Cassidy N, Cunnane G, et al. Quantitative microscopic analysis of inflammation in rheumatoid arthritis synovial membrane samples selected at arthroscopy compared with samples obtained blindly by needle biopsy. Arthritis and Rheumatism 1998;41(4):663–9. [28] Baeten D, Van den Bosch F, Elewaut D, Stuer A, Veys EM, De Keyser F. Needle arthroscopy of the knee with synovial biopsy sampling: technical experience in 150 patients. Clinical Rheumatology 1999;18(6):434–41. [29] Moseley JB, O’Malley K, Petersen NJ, Menke TJ, Brody BA, Kuykendall DH, et al. A controlled trial of arthroscopic surgery for osteoarthritis of the knee. New England Journal of Medicine 2002;347(2):81–8. [30] Van Oosterhout M, Sont JK, Van Laar JM. Superior effect of arthroscopic lavage compared with needle aspiration in the treatment of inflammatory arthritis of the knee. Rheumatology (Oxford) 2003;42(1):102–7. *[31] Tanaka N, Sakahashi H, Sato E, Hirose K, Ishii S. Effects of needle-arthroscopic lavage with different volumes of fluid on knee synovitis in rheumatoid arthritis. Clinical Rheumatology 2002;21(1):4–9. [32] Tanaka N, Sakahashi H, Hirose K, Ishima T, Ishii S. Volume of a wash and the other conditions for maximum therapeutic effect of arthroscopic lavage in rheumatoid knees. Clinical Rheumatology 2006;25(1):65–9. [33] Scire CA, Epis O, Codullo V, Humby F, Morbini P, Manzo A, et al. Immunohistological assessment of the synovial tissue in small joints in rheumatoid arthritis: validation of a minimally invasive ultrasound-guided synovial biopsy procedure. Arthritis Research and Therapy 2007;9(5):R101. *[34] Koski JM, Helle M. Ultrasound guided synovial biopsy using portal and forceps. Annals of the Rheumatic Diseases 2005; 64(6):926–9. [35] Taylor PC. The value of sensitive imaging modalities in rheumatoid arthritis. Arthritis Research and Therapy 2003;5(5):210–3. *[36] Andersen M, Ellegaard K, Hebsgaard JB, Christensen R, Torp-Pedersen S, Kvist PH, et al. Ultrasound colour Doppler is associated with synovial pathology in biopsies from hand joints in rheumatoid arthritis patients: a cross-sectional study. Annals of the Rheumatic Diseases 2013 Mar 8 [Epub ahead of print]. [37] Wilbrink B, van der Heijden I, Schouls LM, van Embden JD, Hazes JM, Breedveld FC, et al. Detection of bacterial DNA in joint samples from patients with undifferentiated arthritis and reactive arthritis, using polymerase chain reaction with universal 16S ribosomal RNA primers. Arthritis and Rheumatism 1998;41(3):535–43. [38] Miossec P, Briolay J, Dechanet J, Wijdenes J, Martinez-Valdez H, Banchereau J. Inhibition of the production of proinflammatory cytokines and immunoglobulins by interleukin-4 in an ex vivo model of rheumatoid synovitis. Arthritis and Rheumatism 1992;35(8):874–83. [39] Bresnihan B, Cunnane G, Youssef P, Yanni G, Fitzgerald O, Mulherin D. Microscopic measurement of synovial membrane inflammation in rheumatoid arthritis: proposals for the evaluation of tissue samples by quantitative analysis. British Journal of Rheumatology 1998;37(6):636–42. [40] Youssef PP, Triantafillou S, Parker A, Coleman M, Roberts-Thomson PJ, Ahern MJ, et al. Variability in cytokine and cell adhesion molecule staining in arthroscopic synovial biopsies: quantification using color video image analysis. Journal of Rheumatology 1997;24(12):2291–8. [41] Kraan MC, Smith MD, Weedon H, Ahern MJ, Breedveld FC, Tak PP. Measurement of cytokine and adhesion molecule expression in synovial tissue by digital image analysis. Annals of the Rheumatic Diseases 2001;60(3):296–8. [42] Dolhain RJ, Ter Haar NT, De Kuiper R, Nieuwenhuis IG, Zwinderman AH, Breedveld FC, et al. Distribution of T cells and signs of T-cell activation in the rheumatoid joint: implications for semiquantitative comparative histology. British Journal of Rheumatology 1998;37(3):324–30. [43] Smeets TJ, Kraan MC, Galjaard S, Youssef PP, Smith MD, Tak PP. Analysis of the cell infiltrate and expression of matrix metalloproteinases and granzyme B in paired synovial biopsy specimens from the cartilage-pannus junction in patients with RA. Annals of the Rheumatic Diseases 2001;60(6):561–5. [44] Kirkham B, Portek I, Lee CS, Stavros B, Lenarczyk A, Lassere M, et al. Intraarticular variability of synovial membrane histology, immunohistology, and cytokine mRNA expression in patients with rheumatoid arthritis. Journal of Rheumatology 1999;26(4):777–84. [45] Tak PP, Smeets TJ, Daha MR, Kluin PM, Meijers KA, Brand R, et al. Analysis of the synovial cell infiltrate in early rheumatoid synovial tissue in relation to local disease activity. Arthritis and Rheumatism 1997;40(2):217–25. [46] Ulfgren AK, Grondal L, Lindblad S, Khademi M, Johnell O, Klareskog L, et al. Interindividual and intra-articular variation of proinflammatory cytokines in patients with rheumatoid arthritis: potential implications for treatment. Annals of the Rheumatic Diseases 2000;59(6):439–47. [47] Klimiuk PA, Goronzy JJ, Bjor nJ, Beckenbaugh RD, Weyand CM. Tissue cytokine patterns distinguish variants of rheumatoid synovitis. American Journal of Pathology 1997;151(5):1311–9. [48] Thurlings RM, Wijbrandts CA, Mebius RE, Cantaert T, Dinant HJ, van der Pouw-Kraan TC, et al. Synovial lymphoid neogenesis does not define a specific clinical rheumatoid arthritis phenotype. Arthritis and Rheumatism 2008;58(6): 1582–9. [49] van de Sande MG, Gerlag DM, Lodde BM, van Baarsen LG, Alivernini S, Codullo V, et al. Evaluating antirheumatic treatments using synovial biopsy: a recommendation for standardisation to be used in clinical trials. Annals of the Rheumatic Diseases 2011;70(3):423–7. *[50] Kraan MC, Haringman JJ, Ahern MJ, Breedveld FC, Smith MD, Tak PP. Quantification of the cell infiltrate in synovial tissue by digital image analysis. Rheumatology (Oxford) 2000;39(1):43–9. [51] Youssef PP, Smeets TJ, Bresnihan B, Cunnane G, Fitzgerald O, Breedveld F, et al. Microscopic measurement of cellular infiltration in the rheumatoid arthritis synovial membrane: a comparison of semiquantitative and quantitative analysis. British Journal of Rheumatology 1998;37(9):1003–7. [52] Cunnane G, Bjork L, Ulfgren AK, Lindblad S, Fitzgerald O, Bresnihan B, et al. Quantitative analysis of synovial membrane inflammation: a comparison between automated and conventional microscopic measurements. Annals of the Rheumatic Diseases 1999;58(8):493–9.
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[53] van der Hall PO, Kraan MC, Tak PP. Quantitative image analysis of synovial tissue. Methods in Molecular Medicine 2007; 135:121–43. [54] Haringman JJ, Vinkenoog M, Gerlag DM, Smeets TJ, Zwinderman AH, Tak PP. Reliability of computerized image analysis for the evaluation of serial synovial biopsies in randomized controlled trials in rheumatoid arthritis. Arthritis Research and Therapy 2005;7(4):R862–7. [55] Boyle DL, Rosengren S, Bugbee W, Kavanaugh A, Firestein GS. Quantitative biomarker analysis of synovial gene expression by real-time PCR. Arthritis Research and Therapy 2003;5(6):R352–60. [56] van der Pouw Kraan TC, van Gaalen FA, Huizinga TW, Pieterman E, Breedveld FC, Verweij CL. Discovery of distinctive gene expression profiles in rheumatoid synovium using cDNA microarray technology: evidence for the existence of multiple pathways of tissue destruction and repair. Genes and Immunity 2003;4(3):187–96. [57] Judex M, Neumann E, Lechner S, Dietmaier W, Ballhorn W, Grifka J, et al. Laser-mediated microdissection facilitates analysis of area-specific gene expression in rheumatoid synovium. Arthritis and Rheumatism 2003;48(1):97–102. [58] Hashimoto A, Matsui T, Tanaka S, Ishikawa A, Endo H, Hirohata S, et al. Laser-mediated microdissection for analysis of gene expression in synovial tissue. Modern Rheumatology 2007;17(3):185–90. [59] Muller-Ladner U, Judex M, Neumann E, Gay S. Laser capture as a tool for analysis of gene expression in inflamed synovium. Methods in Molecular Medicine 2007;135:91–104.
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Best Practice & Research Clinical Rheumatology journal homepage: www.elsevierhealth.com/berh
4
How to perform and analyse synovial biopsies Daniëlle M. Gerlag, MD PhD a, *, Paul P. Tak, MD PhD a, b, c a
Division of Clinical Immunology and Rheumatology, Academic Medical Centre/University of Amsterdam, F4-105, P.O. Box 22700, 1100 DE Amsterdam, The Netherlands b University of Cambridge, Cambridge, UK c GlaxoSmithKline, Stevenage, UK
a b s t r a c t Keywords: Arthritis Synovitis Synovial tissue Synovial biopsy Arthroscopy Synovial biomarkers Digital image analysis
Although most of the rheumatologic diseases can be diagnosed based on clinical examination combined with additional laboratory and radiographic tests, histological examination of synovial tissue may lead to the correct diagnosis and adjustment of therapy when neoplastic or granulomatous disease, deposition disease or infection in spite of negative synovial fluid culture is suspected. For research purposes synovial tissue analysis is used to investigate the pathological changes of the synovium in studies aimed at elucidating the aetiology and pathogenetic mechanisms involved in arthritis. In addition, the use of synovial biomarkers has been shown to be instrumental in the developmental process of new therapeutics. In this chapter, several minimally invasive techniques for acquiring synovial tissue samples, handling of the tissue and the analysis thereof are described. Ó 2013 Elsevier Ltd. All rights reserved.
Synovial biopsies for clinical practice For most rheumatologic diseases, the diagnosis can be made on the basis of clinical examination, routine laboratory tests, radiographic examination and the analysis of synovial fluid if present. Examination of the aspirated fluid from the joint cavity can help to make a distinction between inflammatory and non-inflammatory arthropathies, and when a crystal-induced or bacterial arthritis is suspected, analysis of the synovial fluid is essential in establishing the diagnosis. When synovial fluid cannot be aspirated, however, or in case of unclassified arthritis, suspicion of neoplastic or granulomatous disease, deposition disease or infection in spite of negative synovial fluid * Corresponding author. Tel.: þ31 20 5667765; fax: þ31 20 6919658. E-mail addresses: [email protected] (D.M. Gerlag), [email protected] (P.P. Tak). 1521-6942/$ – see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.berh.2013.03.006
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culture, then histological examination of synovial tissue may lead to the correct diagnosis and adjustment of therapy [1]. Analysis of synovial biopsy samples can assist in the detection of joint infections. In acute bacterial infections, the tissue usually contains many polymorphonuclear leucocytes, and Gram’s staining sometimes reveals bacteria. When blood and synovial fluid cultures are negative, culture of tissue biopsies may still be positive. Using broad-range bacterial primers to analyse and amplify gene coding for ribosomal RNA (16S rRNA) by polymerase chain reaction (PCR) has been successful in demonstrating bacterial species when cultures were negative [2]. Especially when antibiotic treatment has been initiated before culturing synovial fluid and in case of micro-bacterial infections with bacteria that are difficult to culture or grow slowly, this technique can help in establishing the correct diagnosis [3]. Specific infections, such as mycobacterial infections, can lead to specific histological changes in the synovium. Granulomas and acid-fast organisms may be found in these cases. Applying mycobacterial genus-specific PCR on directly isolated DNA extracts from the tissue may help to demonstrate the infectious agent [4]. Appropriate staining may also allow the detection of other microorganisms, such as fungi and spirochaetes (Lyme disease and syphilis). Gout and pseudo-gout can lead to deposits of tophus-like material in the synovial membrane and cartilage. When synovial fluid analysis is repeatedly negative, tissue examination may be of help in diagnosing these deposition diseases. When gout is suspected, the tissue should be conserved in absolute alcohol because the monosodium urate crystals will dissolve in most other fixatives. Unstained sections can be examined with the polarisation microscope, but using DeGolanthal stain for urate is also possible. In amyloidosis, specific amyloid deposits can be found in the synovial tissue. These deposits will colour pink with haematoxylin–eosin and red with Congo red staining and can be surrounded by macrophages, multinucleated giant cells and granulation tissue. Other pigments can be found if appropriate staining is applied, such as in the case of ochronosis, haemochromatosis as well as in recurrent haemarthrosis. In synovial chondromatosis, pigmented villonodular synovitis (PVNS) and multicentric reticulohistiocytosis specific changes of the synovial tissue histology can be found. Other specific changes can be found in the case of synovial chondrosarcomas, synovial haemangiomas, lipoma arborescens and intracapsular chondromas, as well as in Erdheim–Chester disease, sarcoidosis and arthritis caused by foreign-body material [5]. Taken together, examination of synovial tissue may help to make a diagnosis in selected cases of infectious, infiltrative and deposition diseases of joints.
Practice points Synovial tissue analysis may assist in the diagnostic work-up in the case of suspicion of neoplastic or granulomatous disease, deposition disease or infection in spite of negative synovial fluid culture. Synovial biopsies for research purposes Examination of the synovial tissue is also used to investigate the pathological changes of the synovium in studies aimed at elucidating the aetiology and pathogenetic mechanisms involved in arthritis. Descriptive studies of the cell infiltrate in the synovial tissue have provided insight into the pathogenesis and contributed greatly to the understanding of the role of different cell types and various mediators in various forms of arthritis. As a result, this knowledge has led to the identification of potential new targets of therapy for these diseases. Differentiation of early arthritis Although analysis of the synovial tissue could conceivably be helpful in establishing an early diagnosis in patients presenting with immune-mediated arthritis of recent onset, many of the pathological changes found in the inflamed synovium are not specific for any form of arthritis.
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Nonetheless, synovial tissue analysis might have diagnostic potential in distinguishing for instance rheumatoid arthritis (RA) from other forms of arthritis, which would be important in patients presenting with mono- or oligoarthritis, before they fulfil the classification criteria for RA. This is of importance knowing the evidence that early therapeutic intervention with disease-modifying antirheumatic therapies delays or inhibits structural damage in this disease. Being able to use the so-called ‘window of opportunity’ in these patients, while not overtreating other patients who will not develop RA, would be a major step forward towards personalised medicine. From early arthritis clinics it is known that in about 30% of the patients the diagnosis of RA can be established using the 1987 American College of Rheumatology (ACR) criteria at the time of presentation. Another 30% of the patients have an unclassified arthritis [6]. Of these patients, some will develop RA during the follow-up period. Several factors, such as the presence of various autoantibodies, can help to identify the patients who will develop RA using the recently published 2010 American College of Rheumatology/European League Against Rheumatism (ACR/EULAR) criteria, meant to diagnose RA earlier [7,8]. In the synovial tissue, massive infiltration by plasma cells and macrophages in the synovial sublining can predict the diagnosis of RA solely on the basis of examination of synovial biopsy specimens with an accuracy of 85% (Fig. 1). A diagnosis other than RA can be predicted in 97% of the cases when minimal infiltration by these cells is found. These variables were identified using multivariate models in a study of 95 patients with early unclassified arthritis, defined by a disease duration of <12 months [9]. In another study of 71 patients, the intensity of B- and T-cell infiltration and the expression of the av integrin could be used to differentiate patients with RA from those with spondyloarthritis and osteoarthritis [10] (Fig. 2). Other reports have suggested that lining layer depth and the number of CD163positive macrophages might help to distinguish between juvenile-onset spondyloarthritis and other juvenile idiopathic arthritis subsets [11] (Fig. 3). Furthermore, enhanced expression and activation of various protein kinases and factors involved in angiogenesis in the synovial tissue of patients with unclassified arthritis can predict the development of RA and erosive disease in these patients [12]. Taken together, there is some evidence that synovial tissue analysis could help to distinguish between different forms of immune-mediated inflammatory arthritis, but at this moment none of the available tests would justify synovial biopsy to establish the diagnosis of RA or spondyloarthritis in clinical practice. The challenge for the near future will be to explore the value of more sophisticated tools for synovial tissue analysis, such as microarray analysis and proteomics. This approach could provide insight into the pathogenesis of different arthritides as well as distinct subsets of RA leading to common signs and
Fig. 1. CD38 positive plasma cells in the inflamed synovial tissue of an RA patient (100 ).
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Fig. 2. CD 3 positive lymphocytes in the synovial tissue of a patient with active rheumatoid arthritis (100 ).
symptoms [13] and may also lead to the identification of novel diagnostic and prognostic markers that may be used to identify patients who are at risk of having persistent and destructive disease. In the context of personalised medicine, such tools could guide the initiation of tailor-made targeted therapies to prevent autonomous disease progression and irreversible joint damage by early intervention.
The use of synovial biomarkers in the evaluation of novel therapies Synovial biomarkers have been used for various goals, including differential diagnosis, prediction of radiological outcome, evaluation of novel therapies and prediction of clinical response to treatment. At
Fig. 3. CD 55 positive fibroblast like synoviocytes in the inflamed RA synovial tissue (100 ).
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Fig. 4. Minimally invasive needle-arthroscopy of the left knee. Biopsies are taken through an infra-patellar portal and kept on a wet gauze.
present synovial biomarkers are mainly used for analysis on the group level. Conceivably, synovial biomarkers might also be developed in the future to use as surrogate markers to predict outcome in individual patients. Recent work has shown that the use of synovial biomarkers is instrumental in the developmental process of new therapeutics. An advantage is that an early therapeutic effect in the target tissue, the synovial tissue, can be detected in relatively small numbers of patients. Therefore, this approach can be used to screen for potential therapeutic effects, which may accelerate decisions in phase I/II clinical trials. In addition, dose selection may be enhanced before large, conventional clinical trials are conducted. If there is initial proof of concept, such high density of data studies provides the rationale for phase III trials that are necessary to determine whether the biological effects found in these earlier studies translate into clinically meaningful improvement. Furthermore, the description of changes after specific interventions provides insight into the mechanism of action of the treatment, as well as into the role of specific cells and molecules in the pathogenesis of the disease studied, leading to discovery of potential targets for novel therapies. The need for the use of biomarkers in this context is becoming increasingly clear from the large number of compounds in the pipeline of pharmaceutical industry, the increasing difficulty including patients with active arthritis in clinical trials due to the success of available treatment as well as financial and ethical reasons [14]. Early proof-of-concept studies including serial synovial biopsies obviously provide data on the specific mechanism of action of a specific intervention. An example would be the evaluation of B cells in different compartments including the synovium after rituximab treatment [15]. In addition, biomarkers that respond to treatment independent of the specific mechanism of action may be measured. Recent work has shown that it is indeed possible to use such biomarkers [16–18], that serve as a sensitive tool when used for selection purposes in early clinical trials [14]. In RA most of the data are available for the number of the CD68-positive macrophages in the synovial sublining of patients with RA, which has been demonstrated to discriminate between effective treatment on the one hand versus ineffective and placebo treatment on the other [17,18]. It should be noted that it is critical to use standardised and validated techniques to detect and quantify the number of CD68-positive macrophages to get reliable results, but this is clearly feasible. Similarly, it has recently been suggested that the number of CD3-positive T cells distinguishes between effective treatment and placebo treatment in patients with psoriatic arthritis, although the data are still limited [19–21].
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Practice points Synovial tissue analysis could help to distinguish between different forms of immunemediated inflammatory arthritis, but at this moment none of the available tests would justify synovial biopsy to establish the diagnosis in clinical practice. Evaluation of serial synovial biopsies may assist in screening for potential efficacy of novel compounds and provide insight into the mechanism of therapy.
Techniques to obtain synovial tissue Various minimally invasive techniques are being used to access synovial tissue, of which the blind needle and arthroscopic techniques are most common. More recently, ultrasound-guided biopsy techniques have been introduced. All these techniques are generally well tolerated and can be performed safely in an outpatient setting. Blind needle biopsy One of the methods to acquire synovial tissue being described in earlier studies is the blind or closed needle biopsy technique, using the Parker-Pearson needle under local anaesthesia. This needle is a simplified 14-gauge biopsy needle that does not require a skin incision. It is a safe, well-tolerated, technically easy and inexpensive method that can be performed in the outpatient setting. After cleansing, the skin, subcutaneous tissue and joint capsule are anaesthetised using 1–2% lidocaine. Following adequate anaesthesia, the trochar is inserted into the joint and the biopsy needle is introduced into the trochar. Multiple biopsies from different locations throughout the joint can be obtained by changing the angle of the needle. When suction is applied to a Luer-Lock syringe attached to the biopsy needle, the tissue can be obtained more easily and filling the joint cavity with 10–20 ml isotonic saline before introducing the trochar may facilitate the procedure in the case of minimal or no clinical evidence of effusion. This technique is mostly used to biopsy larger joints, such as the knee joint, but its application to other joints, including the ankle, wrist, elbow and shoulder, have also been described. A modified shorter needle can be used to access smaller joints. No complications, such as haemarthrosis, have been reported in a series of >800 samples [22]. Modifications to this technique have been described, for instance, using the Tru-Cut needle. This is an inexpensive, disposable item used for a variety of soft-tissue biopsies that can also be used to retrieve synovial tissue through a procedure similar to that described above. In most cases, this technique yields adequate tissue samples, but it can fail when joints are not swollen. Furthermore, it is usually restricted to larger joints, such as the knee joint, and potential sampling error can be introduced because the operator is not able to visually select the tissue. Another disadvantage of the blind needle biopsy technique is that it is not always possible to obtain adequate tissue samples, especially in the case of biopsy of clinically quiescent joints, for example, after successful therapy. Arthroscopic biopsy Another method to obtain synovial tissue that made its entrance in rheumatology during the 1970s is the use of high-definition, small-bore arthroscopy. Various-sized arthroscopes can be used for different joints. Knee arthroscopy can be performed when a small-bore arthroscope or needlearthroscope, with a diameter ranging from 4.5 to 2.7 mm, is introduced into an infrapatellar skin portal, created after disinfecting the skin and anaesthetising the skin, subcutis and knee joint. For the grasping forceps, a suprapatellar second skin portal is installed. This technique can also be applied
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when biopsy samples of an ankle joint are taken, using a medial and lateral malleolar portal. A small-bore arthroscope of 1.9 mm can be used in the case of even a smaller joint, such as the metacarpophalangeal joint or the wrist, through a radial and ulnar skin portal [23]. Since the introduction of local and regional anaesthetics, this procedure can be performed safely in an office-based setting [24] (Fig. 4). There are several potential advantages of this technique when compared to the blind needle biopsy procedure. One of these advantages is the ability to evaluate the synovium and cartilage macroscopically. Studies have shown that macroscopic signs of inflammation at the biopsy site are positively correlated to the immunohistological features of inflammatory activity in the corresponding tissue sample, although the correlations are only very moderate [25,26]. It is important to realise that it is not possible to predict in a reliable way the microscopic features of the inflamed synovium on the basis of the macroscopic appearance at arthroscopy. Although previous reports suggested that macroscopic scoring systems could be used to distinguish different patient groups, these visual scoring systems still need to be validated. In addition, the evaluation of the articular cartilage by arthroscopy could potentially be used for the evaluation of treatment effects, but this method also requires further standardisation and validation. As mentioned above, another advantage of arthroscopy over blind needle biopsy is the possibility to evaluate smaller joints, such as ankles, wrists and even metacarpophalangeal and proximal interphalangeal joints, using arthroscopes with a narrow diameter. This allows the analysis of synovium in an early stage of disease and in the case of a polyarthritis of smaller joints. The measures of inflammation in tissue samples taken from clinically inflamed joints are generally similar irrespective of being obtained through the blind needle technique or by arthroscopy under visual inspection [27]. However, an important difference between the two methods is that it is always possible to obtain adequate amounts of synovial tissue by arthroscopy, even when the volume of the tissue has decreased following effective treatment. The arthroscopic procedure performed by an increasing number of rheumatologists in an outpatient setting under local or regional anaesthesia is well tolerated by the patients and has a low complication rate. Minimal pain or discomfort during the procedure was reported by 35–36% of the patients in a survey among rheumatologists performing this procedure [28]. Minor complications, such as vasovagal reactions and temporary swelling of the joint, were mentioned in 5–10% of the cases. In a recent survey, in which information of 15,682 arthroscopies performed by rheumatologists was collected, the complication rate of haemarthrosis was 0.9%, deep vein thrombosis 0.2% and wound and joint infection 0.1% [24]. The total number of complications reported was 15.1 per 1000 arthroscopies, which is comparable to the figures reported in the orthopaedic literature. The irrigation volume of the knee joint was positively correlated with the rate of wound infection and the total complication rate, possibly as a result of the extended length of the procedure. However, joint lavage may also have positive effects. Arthroscopic joint lavage In the case of persisting arthritis after needle aspiration and the administration of local corticosteroids, joint lavage through arthroscopy can be considered. Removal of pro-inflammatory and destructive mediators as well as cartilage degradation products is the probable reason for the therapeutic effect seen after this procedure. It should be noted, however, that the effect might differ between different forms of arthritis. Although various uncontrolled, retrospective studies have suggested a beneficial therapeutic effect of joint lavage for osteoarthritis of the knee joint, a randomised, placebocontrolled trial did not show superiority of this procedure compared to placebo [29]. For inflammatory arthritis, such as RA, the efficacy of joint lavage appears more promising. Retrospective analysis of the therapeutic effect of needle aspiration or joint lavage in 50 patients did reveal a superior effect of arthroscopic lavage [30]. This effect seems to be dependent on the volume of physiological fluid used during the procedure, with 5–10 l giving the best results [31,32]. Of importance from the methodological point of view, lavage should be avoided when serial biopsies are taken to evaluate the effects of specific treatments, as the results may be biased due to the anti-inflammatory effect of the lavage procedure.
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Ultrasound-guided synovial biopsy Synovial biopsies will probably increasingly be obtained by means of more recently developed office-based mini-invasive, ultrasound-guided techniques [33]. During this procedure, synovial tissue is acquired percutaneously using a portal and a rigid forceps technique [34]. This approach combines the advantages of being minimally invasive and being able to sample inflamed synovial tissue under indirect visual inspection. Using a multifrequency linear transducer, effusion and synovitis can be identified. The sensitivity can be enhanced applying power-Doppler techniques [35]. Under standard sterile conditions and after infiltrating the skin and subcutaneous tissue with local anaesthetics, a 14gauge needle can be inserted into the designated area under ultrasound guidance. After creating a portal, by means of a flexible wire, followed by a percutaneous sheath, synovial samples can be acquired using a rigid forceps [33,34]. The association of ultrasound-determined pathological synovial blood flow and synovial pathology has been established [36].
Practice points Needle biopsy is simple and cheap. Arthroscopic biopsy is technically more complicated but provides adequate synovial tissue samples, even from clinically uninvolved joints, selected under direct vision from both large and small joints. Synovial biopsies will probably increasingly be obtained by means of more recently developed office-based mini-invasive, ultrasound-guided techniques.
Handling of the tissue Depending on the reason for the biopsy procedure, being diagnostic or for research purposes, the tissue needs to be handled differently. For routine pathological examination by light microscopy, the tissue samples should be fixed in 4% formalin and embedded in paraffin. This can be used for haematoxylin and eosin staining and certain immunohistochemical stainings. When gout is suspected, the synovial tissue should be conserved in absolute alcohol because the monosodium urate crystals will dissolve in most other fixatives. Unstained sections can be examined by polarisation microscopy, but using DeGolanthal stain for urate is also possible. In the case of a suspected infection, the tissue should be kept in suitable culture media. Progress has also been made in the use of PCR on synovial biopsies for detection of bacterial DNA [37]. Here, contaminating DNA needs be avoided and the samples should be snap frozen in liquid nitrogen. For research purposes synovial tissue can be processed for histology, immunohistochemical staining, immunofluorescence, in situ hybridisation, PCR, micro-array, proteomics and cell or tissue culture. Immunohistochemistry can be performed on formalin-fixed, paraffin-embedded material or on samples that were snap frozen in optimal cutting temperature (OCT) embedding medium and stored in liquid nitrogen. Detection of messenger RNA (mRNA) is possible in samples that are snap frozen immediately after the tissue is obtained. It is also possible to culture synovial cell populations and whole biopsy samples in tissue culture media [38]. Tissue heterogeneity and representativeness of biopsy samples Most of the data on this subject have been retrieved from synovial tissue of RA patients. Several studies have suggested a degree of morphological heterogeneity in synovial tissue samples taken from one joint [25]. It is possible, however, to quantify several markers of inflammation in a reliable way by examining only a limited area of tissue [39–41]. For T-cell infiltration and expression of activation antigens in RA synovium, a variance of <10% can be reached when at least six biopsy specimens are examined [42], suggesting that representative data can be obtained when a limited number of biopsy
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samples from different areas within one joint are investigated. When the biopsies are taken form an actively inflamed joint, there is on average no difference in the features of synovial inflammation at the pannus–cartilage junction (where the inflamed synovial tissue invades into the cartilage and underlying bone) compared with other regions of the joint [43,44]. A recent paper described the immunohistochemical assessment of the synovial tissue obtained by ultrasound-guided biopsy from the small joints of nine patients with RA. This study showed that acceptable variance was reached when at least eight tissue samples were obtained from one small hand joint to minimise sampling error [33]. The signs of inflammation in tissue samples taken from inflamed small joints compared to larger joints, such as the knee joint, are generally comparable [23], indicating that the inflammation in one inflamed joint is representative of that in other inflamed joints. By contrast, there is large variability of synovial inflammation between different patients in all phases of the disease [45–48]. It is therefore critical that the number of patients is sufficient when different conditions are being compared. In addition, patient cohorts should be stratified for use of medication and disease activity to allow meaningful conclusions based on the analysis of synovial biopsies.
Practice points Depending on the indication for the biopsy procedure the tissue needs to be handled differently. In order to minimise sampling error, at least six to eight biopsy samples from different areas throughout the joint need to be taken. Stratifying for disease activity and use of medication is essential when the features of synovial inflammation are compared between different patient groups. In RA the inflammation in one inflamed joint is generally comparable to that in other inflamed joints. Analysis of the synovial tissue Depending on the purpose of the analysis of the synovial tissue, different methods can be used. Some of these are briefly discussed in this review article [49]. Quantification of inflammation by histology and immunohistochemistry For quantification of synovial inflammation, three methods have been used. The first method, conventional quantitative analysis by counting cells, is reliable but time consuming. In the evaluation of large series of tissue sections, this is an important disadvantage. As it can be anticipated that the analysis of synovial tissue samples will be increasingly incorporated into clinical trial design, other, less time-consuming methods, need to be applied. A method that is more time efficient is semiquantitative analysis. For instance a 5-point scale (ranging from 0 to 4) by several observers can be used and minor differences between observers can be resolved by mutual agreement [50]. Alternatively, the average score could be calculated. This method has been used in many studies [27,45] and is less sensitive to detect small changes than fully quantitative analysis (by definition) [51]. A third technique, digital image analysis, combines the advantages of these two methods, being both sensitive and time efficient [50,52,53]. We use this computer-assisted approach to evaluate 18 randomly selected high-power fields. By marking the intimal lining layer of the synovial tissue, separate analysis of the intimal lining layer versus the synovial sublining can be performed. In this way, positively stained cells as well as the optical density of the stained area can be measured per square millimetre. This technique has been shown to be reliable for the analysis of the cell infiltrate and the expression of cytokines, matrix metalloproteinases, vascular markers, adhesion molecules and chemokines [41,50]. Inter- and intrarater reliability using intraclass correlations showed good reliability and the measurement of change in total positive cell numbers in synovial tissue can be determined reproducibly for various cell types in RA clinical trials [54].
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Gene expression profiling For gene expression analysis on synovial biopsies, a validated real-time quantitative PCR (Q-PCR) on small synovial tissue samples can be used. This method was developed to complement immunohistochemical analysis and uses a cell-based standard curve, generated with complementary DNA (cDNA) derived from activated peripheral blood mononuclear cells. This approach significantly improved the assay reliability by reducing variation and by simplifying assay development. When this method is applied, six or more synovial tissue samples should be pooled to limit sampling error [55]. A method to analyse not only a selected set of genes but also potentially all genes expressed in the synovial tissue is microarray analysis. Using gene expression profiles determined with cDNA microarray analysis, characterisation of specific signatures can lead us to unravel heterogeneous diseases such as RA and spondyloarthritis. Analysis of the expression profiles with a focus on immune-related genes in synovial tissues from patients with RA revealed considerable variability, resulting in the identification of various molecularly distinct forms of this disease [13,56]. It can be anticipated that studies using this approach will deepen the insight into the molecular mechanisms underlying different pathogenetic subsets of RA and might also facilitate stratification in clinical trials. In addition to gene expression profiling of whole tissue samples, it is also possible to examine specific areas selected in tissue sections by laser-mediated microdissection (LMM) [57]. Target areas of cryosections are cut out using a combination of a microscope and a laser-beam generator. Using a nonfocussed laser beam without direct contact to avoid contamination (a process called laser pressure catapulting), tissue compartments or single viable cells can be isolated. This technique has already been used and validated in several studies in rheumatology [58,59]. Combined with the techniques described above, LMM may be a very interesting and novel way to analyse gene expression in tissues form patients with rheumatological diseases.
Practice points For quantification of synovial inflammation digital image analysis is sensitive and time efficient. Quantitative PCR and various microarray analysis techniques can be used to analyse synovial tissue samples for research purposes.
Conclusion Synovial tissue analysis can be of help in establishing a rheumatological diagnosis and may lead to adjustment of treatment in selected cases in clinical practice. Synovial tissue analysis is especially used for research purposes. It is easily accessible and can be acquired using various minimally invasive techniques in a safe office-based setting. The handling and analysis of the tissue depend on the specific question that needs to be answered. In all cases, standardisation and validation of techniques are critical, but previous work has clearly shown that reliable synovial tissue analysis is feasible and may reveal a wealth of information.
Research agenda develop and determine the value of new tools for synovial tissue analysis; identify novel diagnostic and prognostic markers in the synovial tissue; and analyse synovial tissue in individuals at risk of RA and very early RA.
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