Abstract
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.
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 culture, then histological examination of synovial tissue may lead to the correct diagnosis and adjustment of therapy .
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 . 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 . 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 . 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 . Taken together, examination of synovial tissue may help to make a diagnosis in selected cases of infectious, infiltrative and deposition diseases of joints.
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.
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 . 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 . 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 . In another study of 71 patients, the intensity of B- and T-cell infiltration and the expression of the αv integrin could be used to differentiate patients with RA from those with spondyloarthritis and osteoarthritis ( Fig. 2 ). Other reports have suggested that lining layer depth and the number of CD163-positive macrophages might help to distinguish between juvenile-onset spondyloarthritis and other juvenile idiopathic arthritis subsets ( 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 .
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 symptoms 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 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 .
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 . 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 , that serve as a sensitive tool when used for selection purposes in early clinical trials . 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 . 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 .
- •
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 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.
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.
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 . 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 . 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 . In another study of 71 patients, the intensity of B- and T-cell infiltration and the expression of the αv integrin could be used to differentiate patients with RA from those with spondyloarthritis and osteoarthritis ( Fig. 2 ). Other reports have suggested that lining layer depth and the number of CD163-positive macrophages might help to distinguish between juvenile-onset spondyloarthritis and other juvenile idiopathic arthritis subsets ( 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 .
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 symptoms 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 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 .
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 . 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 , that serve as a sensitive tool when used for selection purposes in early clinical trials . 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 . 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 .
- •
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 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.
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