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Synovial Fluid Collection and Analysis
Synovial Fluid Collection and Analysis
Niels C. Pedersen, D.V.M., Ph.D.*
Synovial fluid analysis is essential to the differential diagnosis of joint disease in the dog. The greatest value of synovial fluid analysis is in determining whether a particular joint disorder is inflammatory or noninflammatory in nature. The presence or absence of synovial inflammation is the single most important differentiating feature of many types of joint disease.
COLLECTION There is a mistaken belief that synovial fluid collection must involve anesthesia or heavy sedation of the patient, as well as surgical preparation and draping of the puncture site. Unfortunately, this erroneous belief has prevented many veterinarians from doing synovial fluid analysis as frequently as it should be done. Joint fluid can usually be collected from the joint of any limb, except the elbow and hip, without sedation or anesthesia. The puncture site does not require elaborate surgical preparation and draping. We clip the hair from over the area, wash the skin with soap and water, and then coat the area with an iodine-based disinfectant. The site is not draped and gloves are not routinely used. Using this simplified procedure, we have had no complications in over 1000 taps. By taking a less cautious approach to joint taps, we find that the clinicians and students will do them more routinely, which considerably enhances the value of the procedure. The question is always asked - when should synovial fluid analysis be done, and which joints should be tapped? As a rule, any dog showing persistent or cyclical fever with generalized stiffness or limb lameness is a candidate for synovial fluid analysis. A single joint tap is occasionally insufficient, and the more joints that are tapped the more *Associate Professor, Department of Medicine, School of Veterinary Medicine. University of California, Davis, California
Veterinary Clinics of North America- Vol. 8, No.3, August 1978
495
496
NIELS
C.
PEDERSEN
likely it is that joint inflammation will be diagnosed. When the animal shows a definite lameness, careful palpation of the lame leg for pain and joint swelling, and analysis of the gait will usually indicate the appropriate joint to tap. As a guide, septic joint disease is often monoarticular or pauciarticular and attacks mainly proximal joints, such as the shoulder, hip, or stifle. The intervertebral disc is another joint that is frequently involved with septic processes. In contrast, immunologic joint disorders are frequently polyarticular or pauciarticular, and infrequently monoarticular. In the polyarticular and pauciarticular forms, the joint inflammation is more severe in distal joints, with the carpus and hock being most consistently involved. The elbow joint is commonly involved in monoarticular immunologic joint disease. Joint taps in dogs are usually done with a l-inch, 22 gauge needle, and a 3 ml syringe. A l-inch, 25 gauge needle is preferrable for very small dogs and cats. A 2-inch or longer needle is sometimes required for the hip joint of larger dogs. Samples of synovial fluid can be put into a tube containing heparin or EDT A, although in most cases the volume of synovial fluid is only a drop or two and can be smeared directly onto a slide or between two coverslips. It is important to remember that all the essential information required can be obtained from a single drop of synovial fluid. This information includes an estimate of the white cell count, a differential count including only mononuclear cells and polymorphonuclear neutrophils, and a special stain for microorganisms. An additional small amount of fluid will usually suffice for bacterial culture. By viewing the fluid as it is slowly allowed to drop from the needle, or as it sits on a slide, an accurate estimate of the color, viscosity, and clarity of the fluid can also be made. Sites
Sites for collection of synovial flu.id in the dog differ somewhat, but we have found the following sites to be the most convenient. The shoulder joint is approached by inserting a needle just anterior to the glenohumeral ligament and directing it slightly downward and posteriorly. It is helpful to maintain the shoulder joint in partial flexion during the procedure. It is important when tapping any joint to release the suction from the syringe before withdrawing the needle. This will prevent unnecessary contamination of the sample with blood from the skin, subcutaneous tissues, and synovium. Fluid collection from the elbow joint usually requires sedation or anesthesia, unless the joint sac is greatly distended. Distension of the joint sac, when it occurs, is usually most noticeable on the medial and lateral surfaces of the joint. When the joint sac is distended, a needle can be easily introduced into the most fluctuant area. If it is not distended, it is difficult to enter the joint either medially or laterally. The needle is inserted downward parallel to the olecranon process on the lateral surface
SYNOVIAL FLUID CoLLECTION AND ANALYSIS
497
of the joint. The needle is angled slightly medially to enter the olecranon fossa. The joint should be maintained in partial flexion during the procedure. The carpal joint can usually be entered without sedation or anesthesia by inserting a needle between any of the palpable intercarpal spaces, or into the medial radiocarpal space. The joint is held in flexion during the procedure. Synovial fluid collection from the hip joint usually requires sedation or anesthesia. The joint is entered by inserting the needle over the greater trochanter and directing it slightly downward and forward. The partially flexed stifle joint is entered by inserting the needle into the joint space just lateral to the straight pateller ligament. The needle is directed medially and slightly upward into the area in which the cruciate ligaments originate and insert between the femoral condyles. The hock joint is entered by inserting a needle under the lateral malleolus as it lips over the tibial and fibular tarsal bones. Care is taken to avoid the small superficial vein that runs in the area. The hock joint is usually held in partial flexion during the procedure.
ANALYSIS Gross Appearance The following things are usually noted grossly when the fluid is collected: volume, color, turbidity, and viscosity. It is difficult to obtain more than 0.05 to 0.3 ml of synovial fluid from normal joints. An increased volume of fluid can be seen in degenerative joint disease, especially when an abnormal joint is acutely traumatized, and in inflammatory joint disorders. Even when the joint is inflamed, however, the amount of synovial fluid is often small. Synovial fluid is usually clear and free of blood .. A bloody tap can usually be distinguished because the blood is not completely mixed with the fluid. Blood can sometimes be aspirated from inflamed joints, especially if they are septic or acutely traumatized. A yellow-tinged fluid indicates that hemorrhage into the synovial membrane has occurred, and that hemoglobin pigments are being released into the fluid. This can occur in degenerative joint disease and traumatic joint disease, as well as in inflammatory joint disorders. Normal synovial fluid is usually crystal clear, unless it contains red or white cells in excess numbers. Synovial fluid is normally very viscous in nature. It forms a small glob when placed on a slide and a long string when dropped slowly from the needle tip. A thin, runny consistency indicates that the fluid is deficient in polymerized hyaluronic acid, or that the fluid is greatly diluted with serum. A thin fluid is frequently seen in inflamed joints, and occasionally in degenerative or traumatized joints.
498
NIELS
C.
PEDERSEN
Histological Appearance Normal synovial fluid contains less than 3000 cells per mm. 3 Red blood cells are present in very low numbers in properly collected samples from normal joints. Absolute cell counts are often not done because the volume of synovial fluid is often insufficient. If cell counts are done with a hematocytometer, it is important that the fluid be diluted in physiological saline rather than the normal diluting fluid, which will precipitate the mucin. In cases in which the volume of synovial fluid is very small, cell counts are estimated by noting the number of white cells per microscopic field on a stained smear and comparing this to a blood smear of known concentration. As an example, if a sample of synovial fluid contains 10 white blood cells per field, and a smear from blood with a cell count of 20,000 per mm 3 has 5 white blood cells per field, then the synovial fluid contains approximately 40,000 white blood cells per mm. 3 With practice, even this approximation is not necessary, and it is sufficient to note that the white cell count is normal, mildly elevated, or greatly elevated. Normal synovial fluid contains a mixture of small and large mononuclear cells. These cells are often bizarrely shaped and frequently contain many vacuoles and granules. They can be subclassified into various types, although there is very little value in doing so. The absolute number of mononuclear cells varies greatly, which limits its diagnostic value. There is a tendency, however, for the number of these cells to be increased in traumatized or degenerative joints, joints with osteochondrosis, and chronically inflamed joints. Polymorphonuclear neutrophils are usually absent from normal synovial fluid, or if present, they should not make up more than 5 per cent of the cell population. An increase in the relative proportion and absolute number of polymorphonuclear neutrophils indicates inflammation of the synovial lining. As a rule, the more inflamed the synovium, the greater the concent~ation of white cells in the synovial fluid, and the greater the proportion of white cells that will be neutrophils. The presence of polymorphonuclear neutrophils in the synovial fluid indicates inflammation, but has no differentiating value beyond that. That is to say, the same type of fluid can be seen in any inflamed joint, regardless of the etiology. In addition to the routine white cell count and differential, there are several other cellular phenomena that will occasionally prove helpful in differentiating joint disorders. Lupus erythematosus (LE) cells will occasionally be seen in synovial fluid in cases of canine systemic lupus erythematosus. Sy.novial cells can also be analyzed for gamma G immunoglobulin (IgG) and C3 inclusions. To do this, white cells are centrifuged free of the synovial fluid, washed several times in physiologic saline, and resuspended in a drop of 5 per cent bovine serum albumin in saline. Slide smears are made and fixed for several min-
SYNOVIAL FLUID CoLLECTION AND ANALYSIS
499
utes in absolute acetone. The fixed smears are then reacted with fluorescein isothiocyanate conjugated anticanine IgG or C3 globulin. The presence of IgG, and less frequently C3, containing inclusions in synovial fluid cells is commonly seen in canine erosive (rheumatoid-like) polyarthritis. The are usually absent from the fluids of other inflammatory joint disorders of the dog.
MICROORGANISMS
Contrary to common belief, if a joint is infected, the offending organism can usually be cultured or visualized. Needless to say, success depends largely on obtaining fresh specimens, the expertise of the person culturing the fluid, the choice of growth media, and the person's familiarity with identifying organisms in special stained smears. Certain organisms, such as mycoplasma or bacterial L-forms, require specialized stains and growth media to visualize them or to grow them in vitro.
SUMMARY In conclusion, synovial fluid analysis in the dog has two major purposes: to determine whether the synovial membrane is inflamed, and to visualize or isolate microorganisms. These two facts, coupled with the clinical history, and physical and radiographic findings, are extremely important in determining the etiology of the joint disorder. Department of Medicine School of Veterinary Medicine University of California Davis, California 95616
Niels C. Pedersen, D.V.M., Ph.D.*
Synovial fluid analysis is essential to the differential diagnosis of joint disease in the dog. The greatest value of synovial fluid analysis is in determining whether a particular joint disorder is inflammatory or noninflammatory in nature. The presence or absence of synovial inflammation is the single most important differentiating feature of many types of joint disease.
COLLECTION There is a mistaken belief that synovial fluid collection must involve anesthesia or heavy sedation of the patient, as well as surgical preparation and draping of the puncture site. Unfortunately, this erroneous belief has prevented many veterinarians from doing synovial fluid analysis as frequently as it should be done. Joint fluid can usually be collected from the joint of any limb, except the elbow and hip, without sedation or anesthesia. The puncture site does not require elaborate surgical preparation and draping. We clip the hair from over the area, wash the skin with soap and water, and then coat the area with an iodine-based disinfectant. The site is not draped and gloves are not routinely used. Using this simplified procedure, we have had no complications in over 1000 taps. By taking a less cautious approach to joint taps, we find that the clinicians and students will do them more routinely, which considerably enhances the value of the procedure. The question is always asked - when should synovial fluid analysis be done, and which joints should be tapped? As a rule, any dog showing persistent or cyclical fever with generalized stiffness or limb lameness is a candidate for synovial fluid analysis. A single joint tap is occasionally insufficient, and the more joints that are tapped the more *Associate Professor, Department of Medicine, School of Veterinary Medicine. University of California, Davis, California
Veterinary Clinics of North America- Vol. 8, No.3, August 1978
495
496
NIELS
C.
PEDERSEN
likely it is that joint inflammation will be diagnosed. When the animal shows a definite lameness, careful palpation of the lame leg for pain and joint swelling, and analysis of the gait will usually indicate the appropriate joint to tap. As a guide, septic joint disease is often monoarticular or pauciarticular and attacks mainly proximal joints, such as the shoulder, hip, or stifle. The intervertebral disc is another joint that is frequently involved with septic processes. In contrast, immunologic joint disorders are frequently polyarticular or pauciarticular, and infrequently monoarticular. In the polyarticular and pauciarticular forms, the joint inflammation is more severe in distal joints, with the carpus and hock being most consistently involved. The elbow joint is commonly involved in monoarticular immunologic joint disease. Joint taps in dogs are usually done with a l-inch, 22 gauge needle, and a 3 ml syringe. A l-inch, 25 gauge needle is preferrable for very small dogs and cats. A 2-inch or longer needle is sometimes required for the hip joint of larger dogs. Samples of synovial fluid can be put into a tube containing heparin or EDT A, although in most cases the volume of synovial fluid is only a drop or two and can be smeared directly onto a slide or between two coverslips. It is important to remember that all the essential information required can be obtained from a single drop of synovial fluid. This information includes an estimate of the white cell count, a differential count including only mononuclear cells and polymorphonuclear neutrophils, and a special stain for microorganisms. An additional small amount of fluid will usually suffice for bacterial culture. By viewing the fluid as it is slowly allowed to drop from the needle, or as it sits on a slide, an accurate estimate of the color, viscosity, and clarity of the fluid can also be made. Sites
Sites for collection of synovial flu.id in the dog differ somewhat, but we have found the following sites to be the most convenient. The shoulder joint is approached by inserting a needle just anterior to the glenohumeral ligament and directing it slightly downward and posteriorly. It is helpful to maintain the shoulder joint in partial flexion during the procedure. It is important when tapping any joint to release the suction from the syringe before withdrawing the needle. This will prevent unnecessary contamination of the sample with blood from the skin, subcutaneous tissues, and synovium. Fluid collection from the elbow joint usually requires sedation or anesthesia, unless the joint sac is greatly distended. Distension of the joint sac, when it occurs, is usually most noticeable on the medial and lateral surfaces of the joint. When the joint sac is distended, a needle can be easily introduced into the most fluctuant area. If it is not distended, it is difficult to enter the joint either medially or laterally. The needle is inserted downward parallel to the olecranon process on the lateral surface
SYNOVIAL FLUID CoLLECTION AND ANALYSIS
497
of the joint. The needle is angled slightly medially to enter the olecranon fossa. The joint should be maintained in partial flexion during the procedure. The carpal joint can usually be entered without sedation or anesthesia by inserting a needle between any of the palpable intercarpal spaces, or into the medial radiocarpal space. The joint is held in flexion during the procedure. Synovial fluid collection from the hip joint usually requires sedation or anesthesia. The joint is entered by inserting the needle over the greater trochanter and directing it slightly downward and forward. The partially flexed stifle joint is entered by inserting the needle into the joint space just lateral to the straight pateller ligament. The needle is directed medially and slightly upward into the area in which the cruciate ligaments originate and insert between the femoral condyles. The hock joint is entered by inserting a needle under the lateral malleolus as it lips over the tibial and fibular tarsal bones. Care is taken to avoid the small superficial vein that runs in the area. The hock joint is usually held in partial flexion during the procedure.
ANALYSIS Gross Appearance The following things are usually noted grossly when the fluid is collected: volume, color, turbidity, and viscosity. It is difficult to obtain more than 0.05 to 0.3 ml of synovial fluid from normal joints. An increased volume of fluid can be seen in degenerative joint disease, especially when an abnormal joint is acutely traumatized, and in inflammatory joint disorders. Even when the joint is inflamed, however, the amount of synovial fluid is often small. Synovial fluid is usually clear and free of blood .. A bloody tap can usually be distinguished because the blood is not completely mixed with the fluid. Blood can sometimes be aspirated from inflamed joints, especially if they are septic or acutely traumatized. A yellow-tinged fluid indicates that hemorrhage into the synovial membrane has occurred, and that hemoglobin pigments are being released into the fluid. This can occur in degenerative joint disease and traumatic joint disease, as well as in inflammatory joint disorders. Normal synovial fluid is usually crystal clear, unless it contains red or white cells in excess numbers. Synovial fluid is normally very viscous in nature. It forms a small glob when placed on a slide and a long string when dropped slowly from the needle tip. A thin, runny consistency indicates that the fluid is deficient in polymerized hyaluronic acid, or that the fluid is greatly diluted with serum. A thin fluid is frequently seen in inflamed joints, and occasionally in degenerative or traumatized joints.
498
NIELS
C.
PEDERSEN
Histological Appearance Normal synovial fluid contains less than 3000 cells per mm. 3 Red blood cells are present in very low numbers in properly collected samples from normal joints. Absolute cell counts are often not done because the volume of synovial fluid is often insufficient. If cell counts are done with a hematocytometer, it is important that the fluid be diluted in physiological saline rather than the normal diluting fluid, which will precipitate the mucin. In cases in which the volume of synovial fluid is very small, cell counts are estimated by noting the number of white cells per microscopic field on a stained smear and comparing this to a blood smear of known concentration. As an example, if a sample of synovial fluid contains 10 white blood cells per field, and a smear from blood with a cell count of 20,000 per mm 3 has 5 white blood cells per field, then the synovial fluid contains approximately 40,000 white blood cells per mm. 3 With practice, even this approximation is not necessary, and it is sufficient to note that the white cell count is normal, mildly elevated, or greatly elevated. Normal synovial fluid contains a mixture of small and large mononuclear cells. These cells are often bizarrely shaped and frequently contain many vacuoles and granules. They can be subclassified into various types, although there is very little value in doing so. The absolute number of mononuclear cells varies greatly, which limits its diagnostic value. There is a tendency, however, for the number of these cells to be increased in traumatized or degenerative joints, joints with osteochondrosis, and chronically inflamed joints. Polymorphonuclear neutrophils are usually absent from normal synovial fluid, or if present, they should not make up more than 5 per cent of the cell population. An increase in the relative proportion and absolute number of polymorphonuclear neutrophils indicates inflammation of the synovial lining. As a rule, the more inflamed the synovium, the greater the concent~ation of white cells in the synovial fluid, and the greater the proportion of white cells that will be neutrophils. The presence of polymorphonuclear neutrophils in the synovial fluid indicates inflammation, but has no differentiating value beyond that. That is to say, the same type of fluid can be seen in any inflamed joint, regardless of the etiology. In addition to the routine white cell count and differential, there are several other cellular phenomena that will occasionally prove helpful in differentiating joint disorders. Lupus erythematosus (LE) cells will occasionally be seen in synovial fluid in cases of canine systemic lupus erythematosus. Sy.novial cells can also be analyzed for gamma G immunoglobulin (IgG) and C3 inclusions. To do this, white cells are centrifuged free of the synovial fluid, washed several times in physiologic saline, and resuspended in a drop of 5 per cent bovine serum albumin in saline. Slide smears are made and fixed for several min-
SYNOVIAL FLUID CoLLECTION AND ANALYSIS
499
utes in absolute acetone. The fixed smears are then reacted with fluorescein isothiocyanate conjugated anticanine IgG or C3 globulin. The presence of IgG, and less frequently C3, containing inclusions in synovial fluid cells is commonly seen in canine erosive (rheumatoid-like) polyarthritis. The are usually absent from the fluids of other inflammatory joint disorders of the dog.
MICROORGANISMS
Contrary to common belief, if a joint is infected, the offending organism can usually be cultured or visualized. Needless to say, success depends largely on obtaining fresh specimens, the expertise of the person culturing the fluid, the choice of growth media, and the person's familiarity with identifying organisms in special stained smears. Certain organisms, such as mycoplasma or bacterial L-forms, require specialized stains and growth media to visualize them or to grow them in vitro.
SUMMARY In conclusion, synovial fluid analysis in the dog has two major purposes: to determine whether the synovial membrane is inflamed, and to visualize or isolate microorganisms. These two facts, coupled with the clinical history, and physical and radiographic findings, are extremely important in determining the etiology of the joint disorder. Department of Medicine School of Veterinary Medicine University of California Davis, California 95616