Synovial Fluid Analysis
Synovial fluid is a thick liquid found in the joints formed at the end of two long bones. It is also called joint fluid. The purpose of this fluid is to serve as a cushion between the bones and reduce friction when motion is initiated. Synovial fluid analysis is required when a practitioner needs to diagnose a variety of disorders. Such exam will allow the clinician to check on the qualities of the fluid namely the color or the thickness. Chemical studies can also be performed to appreciate the viscosity and any change in chemicals. Finally, a microscopic analysis of the fluid is also possible to look for bacteria, or crystals or any other substances.
The synovial fluid analysis helps diagnose the cause of joint pain or inflammation in response to an injury or an infection or other diseases. Clinically, pain, redness of the joint swelling and inflammation with the loss of range of motion may be as well present. A joint effusion can be seen in many occasions and most commonly in any form of arthritis especially jn Osteoarthritis which is a degenerative arthritis seen as the joint ages with time. It is generally a chronic and progressive process of deterioration of the articular surface rendering the articulation intermittently painful or deformed with cartilage breakdown, leading to a loss of motion.
In Rheumatoid arthritis, the body immune system attacks the healthy cells in the joints. While in Gouty arthritis, there is an inflammatory process in one or more joints but usually the great toe is involved. A joint effusion is almost always present when infection is suspected. In disease like hemophilia, an inherited disorder, excessive bleeding in a closed joint space, is often seen because of synovial bleeding.
Whenever a patient presents self with a joint pain or some swelling or redness, the clinician may choose to perform a joint aspiration to asset a diagnosis of infection, or in case of acute gout etc. The synovial fluid is collected via arthrocentesis or joint aspiration with proper asepsis of the affected joint. An anesthetic agent like Zylocain or Lidocaine can be injected to numb the skin prior to the procedure but it may become difficult with a child or an adult afraid of the needle for which a light sedation can be given prior to the aspiration. This may reduce anxiety calming the individual prior to the procedure. This will allow the provider to perform a less than two minutes procedure and to withdraw a sample of fluid into a syringe for further analysis and diagnosis. The point of aspiration is generally covered with a band-aid dressing.
Occasionally the practitioner may require avoiding eating or drinking before the test or you may receive specific instructions to follow. You may be sore for a couple of hours or days after the aspiration. Generally, it is not expected that complications will happen after such a procedure but rarely secondary infections may be possible or a recurrent collection or a collection of fluid especially blood can be seen.
In sport Medicine, it is proven that gross mechanical abnormalities contribute to posttraumatic osteoarthritis and recent researches have demonstrated that the damage to the articular cartilage may contribute to changes in the joint microenvironment. Biomarkers have been discovered exhibiting an altered expression in the synovial fluid after knee injury and for years those molecules remain elevated in the synovial fluid. These biomarkers have the potential to aid in the early detection of post traumatic osteoarthritis before even radiologic evidence can become apparent. More, deciphering the process at the micro-articular level after an episode of trauma may certainly allow a better and earlier identification of therapeutic targets in the prevention of osteoarthritis.
All sports contribute to knee injuries. Meniscal tears, Cruciate ligaments tears and osteochondral defects are seen in more than half of athletes after a knee injury. These injuries contribute to the development of post traumatic osteoarthritis. Alterations in the joint mechanism and the range of motion certainly alter the distribution of the load across the articular surface to precipitate changes and later progression to osteoarthritis. Meniscal tears and Anterior cruciate ligaments are common and the O’Donoghue triad is a well-known entity feared by all orthopedic surgeons amplifying changes in the joint because of the loss in stability and the lack of chondroprotection. It remains a puzzle to us to appreciate these changes in younger patients.
Prior studies have identified the existence of cytokines, chemokines with growth factors responsible for the changes with an altered expression in the synovial fluid after knee injury. These molecules remain elevated in the traumatized joints for years and new researches found them implicated in the genesis of early osteoarthritis. These factors may serve as a useful diagnostic and prognostic biomarkers after trauma. The Academy of Orthopaedic Surgeons (AAOS) has shown an interest in identifying the Osteoarthritis markers as part of an on-going investigation to target biologic markers and their relationship with traumatic injury to the knee.
Let us review some biomarkers (biologic markers):
1- Biomarkers like Cytokines (Il-6) are secreted by T-cells and Microphages during an infectious process or after a trauma to a joint. They will also stimulate IL-10-1Ra production to modulate the inflammatory response.
2- INF alpha is a macrophage derived cytokine that stimulate the production of cytokines from the chondrocytes (IL-1) and metalloproteinase synthesis from the synovial cells, suppressing the synthesis of cartilage proteoglycans. They function like the cytokines as discussed above.
3- Chemokines (Il-8) a chemokine that recruits neutrophils in the site of infection.
4- MIP Chemokine produced also by the neutrophils, activates the granulocytes and also recruits the neutrophils, monocytes, macrophages, the immature dendritic cells and Th1 cells (helper cells) to the site of inflammation.
5- MCP (Monocyte chemotactic protein) Chemokine recruits’ monocytes and macrophages to the sites of the infection.
6- CXCL6/GCP2 (Chemokine ligand and Granulocyte chemotactic protein) Chemokine recruits’ neutrophils also to the site of the inflammation.
7- The Growth Factors VEGF (vascular endothelial growth factor) induces angiogenesis.
8- bFGF (Basic fibroblast growth factor) promotes chondrogenesis, angiogenesis, wound healing and formation of granulation tissue.
9- Ghrelin stimulates osteoblast and serve as a growth factor for chondrocytes; They imbibes a number of inflammatory cytokines including Il-6 and TNF-alpha (Tumor Necrosis Factor)
10- The catabolic inhibitors MMP (matrix metalloproteinase) and TIMP (tissue inhibitor of metalloproteinase) are a group of endopeptidases that degrade extracellular matrix in psychological tissue remodeling and stimulating embryogenesis.
11- TIMP is a class of proteins that regulate MMPs endopeptidases and to promote cell growth of chondrocytes. They do have as well a protective effect.
Interleukin 6 is a cytokine that stimulates synoviocytes, meniscal chondrocytes and articular chondrocytes to express catabolic enzymes involved in the cartilage degradation. It is well accepted that the iL-6 levels are increased in ACL-knee deficient when compared to the opposite knee. But it is less clear that Il-6 increases when the meniscus is torn. In vitro, IL6 is associated with cartilage degradation by example in mouse models, inhibition of IL-6 has been shown to alleviate cartilage lesions and impair osteophyte formation like seen in post-traumatic osteoarthritis after a medial meniscal injury. It looks like iL-6 is a predictor of cartilage damage with a degree of chondral degeneration.
As already stated, IL6 does not appear to be significantly elevated in patients with a meniscal tear and an ACL injury compared to a meniscal tear alone (Isolated ACL tear) and no osteoarthritis as reported by Ding. He found two biomechanical and distinct patterns. IL6 appears to be higher in women than men as well as in symptomatic persons with older age. There is an association between IL6 and pain and IL6 appears to act on the nociceptors of the animal model knees. Time elapsed between injury and surgical treatment could also contribute to the production of IL6 in the synovial fluid after meniscectomy. Still a study among patient with chronic meniscal tear showed elevation also of the IL-6 even one year post-operatively compared to the general population.
IL-8 is expressed by articular cartilage in response to a number of stimuli including lipopolysaccharide (LPS), tumor necrosis factor-alpha (TNF-alpha) and IL-1 beta expression. IL-8 has been implicated as a main factor involved in neutrophil chemotaxis in the joint. IL-8 messenger RNA is upregulated in patients undergoing meniscectomy for long-standing meniscal tears especially following synovitis. Other authors like Liu and al found that IL-8exhibits a lower expression in complex meniscal tears by presenting more sulfated glycosaminoglycans (sGAG), a cartilage breakdown product. Many other papers echoed the correlation with different breakdown products like the C2C found in early osteoarthritis. Further research is needed. To understand the role of IL-8 in the formation of osteoarthritis.
Macrophages Inflammatory Proteins (MIPs) are released by the neutrophiles to recruit immune cells in infection and inflammation: MIP-1 alpha (chemokine ligand-3) found in patients with ACL injury combined with meniscal tear rather than meniscal tear alone. Nair and al found also other markers MIP -3 Betha or CCL19.
Monocyte Chemotactic Proteins (MCP’s) are key initiators of inflammation that trigger chemotaxis and the migration of monocytes and macrophages to the sites of inflammation by interreacting with the membrane receptor C-C chemokine receptor type 2 (CCR2). It seems that the MCP’s play a role in chondrocytes apoptosis, inhibition of chondrocyte proliferation and chondrocyte stimulation to increase the expression of matrix metalloproteinases (MMPs). MCP-1CCp was also found in to be elevated in knees with meniscal tear or ACL tear. It is found also with old age and it seems that the MCP-1 may play a role in the production of pain similarly to IL-6.
Chemokine Ligan 6 (CXCL6) are granulocytes chemotactic protein 2 (GCP2) which mediates neutrophile chemotaxis by activating 2 related proteins: CXCR1 and CXCR2, Brophy noted that a substantial level of Chemokine Ligan 6 in the joint, especially in the presence of traumatic tears when compared to degenerative tears, indicating a distinction in relation to the microenvironment.
MMPs are a group of endopeptidases that degrade the extracellular matrix for remodeling and embryogenesis. MMP1 degrades in collagen type 1 and this is the main component of the meniscal matrix. There is a direct relation between the MMP’s and the extracellular matrix. MMP is known to be released in static compression suggesting that inflammation in the joint results in a cascade of MMP activation and ongoing cartilage regeneration. MMP level is increased 25-fold in the synovial fluid of a knee with a meniscal tear. MMP correlate positively with cartilage strain especially when the knee is in maximum flexion. It is also proportional to the post-operative pain level. Others have found simply an elevation of the mmP-3 in synovial fluid of knees with meniscal tears (Clair et al) while Liu did not but found the MMP-10 to be decreased. MMP-1 and MMP-3 were noted to be increased in traumatic meniscal tears but not in degenerative tears.MMP-13 showed higher level in patients with combined ACL and meniscal tears compared to isolated meniscal tear.
So, in an ACL injury, the MMP-3 and the TIMP-1 were both elevated and the MMP-3 remained elevated independently to the duration of the injury. An elevation in the MMP-9 was also noted suggesting a poor outcome following surgical stabilization. It remains unclear that MMP and TIMP can predict post traumatic osteoarthritis 15 years later. Just on the basis of the clinical implications after an ACL injury.
TNF alpha is a macrophage derived cytokine which has for function to stimulate the secretion of cytokines from the chondrocytes.in increasing the IL-1 and the metalloproteinase synthesis from the synovial cells. It also suppresses the synthesis of the cartilage proteoglycans. TNF alpha becomes 6-fold elevated but over the next five years the concentration diminishes over the next five years because of the inflammatory process. Others have shown no difference even when arthritic changes were present
Vascular endothelial Growth Factor (VEGF) is an angiogenesis factor that is expressed by hypertrophic chondrocytes acting like osteoblasts, endothelial cells or other chondrocytes occasionally inhibiting aggrecan and collagen II synthesis. This process works primarily in the chemotaxis of the endothelial cells inhibiting aggrecan and collagen II synthesis. It may be associated to the severity of the osteoarthritis. Some in-vivo studies have demonstrated that any primary human chondrocytes incubated in the synovial fluid of a patient with osteoarthritis, showed an elevation of the VEGF-positive chondrocytes. The same observations were made in meniscus -deficient knees and the presence of high-grade cartilage lesions. So, patients with meniscus deficient knees and Osteoarthritis
Ghrelin is a gastric derived hormone which stimulated osteoclasts in the musculoskeletal system and serves as a growth factor for chondrocytes. It plays also a role in the inflammation process in inhibiting the IL-6 and TNF-alpha. Recent studies using mouse models of post traumatic osteoarthritis (Qu and al), exogenous ghrelin downed regulated key cytokines, inhibited chondrocytes apoptosis in lowering the MMP level.in the joint. To-date more studies have demonstrated an independent but negative association with meniscal injury or cartilage damage in ACL deficient models. For these authors, Ghrelin may delay cartilage degeneration.
Fibroblast growth factor (bFGF) promotes numerous cell functions including angiogenesis, cell proliferation, wound healing and tissue remodeling. They represent the main mediator for maintaining cartilage homeostasis by stimulating or stabilizing chondrocytes production of extracellular matrix components. FGF also play a role in tendon repair by increasing collagen III production and cell proliferation but in ACL tears with no cartilage damage, there is a lower concentration of FGF-2.
In conclusion, any knee injury may increase the risk for post traumatic osteoarthritis (PTOA) and the clinical diagnosis remains reliant on a clinical history of pain with functional decline, instability and later radiologic changes. As presented above, there is now growing evidence that many biomechanical factors may play a role in the process even before any evidence of radiologic changes can be detected. The analysis of the synovial fluid should point toward a diagnosis of osteoarthritis or rheumatoid arthritis or even gout. It may point toward a bleeding disorder or a bacterial infection. More we have presented different biologic factors available to help in the diagnosis of post-traumatic osteoarthritis. This is a new adventure in research for the orthopedic surgeons and I wish that every orthopedist and or other healthcare providers can become familiar with the changes so they can certainly sharpen their skills. This is a new field of expertise and one will have to learn a lot to gain proper expertise.
Maxime J-M Coles MD
Boca Raton FL
1- Carbone A, Rodeo S: Review of current understanding of post-traumatic osteoarthritis resulting from sport injuries. J Orthop Res 2017 Mar, 35 (3): pp 397-405.
2- Majewski M, Susanne H, Klaus S. Epidemiology of athletic knee injurie: A 10=year study. Knee 2006 Jun; 13 (3): pp 184-188