49

Synovial fluid

49

Synovial fluid

49

Synovial fluid

49

Synovial fluid

  49 Synovial fluid

Gerald Partsch, Mirjam Franz, Rudolf Gruber, Lothar Thomas

Usually, a healthy synovial fluid is considered as the best lubricant to the natural hip or knee joints. It provides a nearly frictionless movement and acts as a shock absorber and plays an important role in the healing process after surgery. Articular cartilage works as a lubricant cavity and offers its role, along with synovial fluids, as the boundary lubrication, while the water component of the synovial fluid squeezes out /1/.

Synovial fluid (SF) analysis is of interest for the rheumatologist, orthopedic surgeon, and physician because numerous biochemical, cytological, and immunological SF alterations occur in many rheumatic diseases.

The clinical value of SF analysis is controversial but its role as an additional diagnostic aid has undoubtedly been recognized. The examination of SF can be used as the sole diagnostic criterion only in cases of gout, pseudo-gout, other crystal depositions, and septic arthritis /2/. Other authors /3/ are of the opinion that a thorough examination, even of effusions involving small joints, should be carried out on a routine basis in all patients with joint diseases. This would allow the diagnosis of gout or pseudo-gout also in asymptomatic joints.

The main goal of SF examination is to connect with a corresponding joint condition to appropriate underlying disorder and to rule out other diagnoses. Frequently it is shown that presumed degenerative changes associated with a decline in functional range of motion are also accompanied by an inflammatory process.

Refer to Tab. 49-1 – Diseases with joint effusions and their classification into four major categories.

49.1 Indication

Differentiation between acute synovitis (sepsis, crystals) and chronic arthropathy /4/.

Chronic active inflammation in patients with failed joint replacements undergoing revision surgery /5/.

49.2 Sampling of synovial fluid

While obtaining a sample of SF bacterial contamination of the generally sterile joint must be avoided. For details on arthrocentesis of different joints refer to reference /4/.

49.2.1 Knee joint aspiration

The patient is in a supine position with completely extended knee. After joint palpation the area posterior to the medial patellar edge is marked. After cleansing and disinfection of the skin surface overlying the joint the needle is introduced, using local anesthesia, into the joint space at a slightly downward angle and SF is aspirated.

Materials required

Alcohol swabs, disinfectant (iodized), gauze, sterile 3, 10, and 20 mL syringes with 18-gauge × 1 ½-inch needles for viscous effusions (purulent or inclusion bodies), 20-gauge × 1 ½-inch needles for the knee joint, and 25-gauge × 5/8-inch needles for small joints; sterile single use collection tubes (10 mL) containing sodium heparin; clean microscope slides for microscopy and cover glass; possibly appropriate culture medium for bacteriological examination; local anesthetic agents.

Problems

In the case of suspected infectious arthritides sterile physiological saline can be injected if no SF can be obtained during the joint tap followed by bacteriological examination of the aspirated fluid.

Error during joint aspiration

The are the following contra indications /4/:

  • Injection to cellulitic or broken skin
  • Patients who are unstable coagulated
  • A prosthetic joint should not be aspirated without prior consultation with an orthopedic surgeon or rheumatologist.

Pre analytical points

Important pre analytical points are:

  • When obtaining SF sodium heparin should be used as the anticoagulant because the use of lithium heparin or oxalate can result in the formation of SF crystals causing false positive results during the microscopic evaluation
  • When there is less than 1 mL of SF the addition of liquid anticoagulants can result in the destruction of cellular components
  • Improper aspiration technique can result in damage to blood vessels and thus blood stained SF.

49.3 Basic investigations of the synovial fluid

Synovial fluid analysis includes the following diagnostic tests /67/ (Tab. 49-2 – Examinations for synovial fluid analysis):

  • Basic examinations (viscosity, mucin content, and pH), not requiring special aids, allowing a preliminary assessment of which disease category the SF can be assigned to (Tab. 49-1 – Diseases with joint effusions and their classification into four major categories)
  • Hematological investigations and inflammation markers to determine the degree of inflammation and in some cases the cause
  • Crystal identification for diagnosis of crystal arthritis
  • Microbiological examinations for diagnosis of septic arthritis
  • Biochemical and immunological investigations which may be of some clinical relevance

In Fig. 49-1 – Algorithm for synovial fluid investigation a schematic approach to the examination is suggested.

49.3.1 Volume

The volume of joint fluid is determined following arthrocentesis. It is known from measurements made involving knee joints of deceased number of persons that the volume of SF in the healthy joint is up to about 3.5 mL /3/. The total volume of SF is not obtained during regular arthrocentesis but this is not required for a valid diagnostic assessment. More than 3.5 mL of SF can be considered as an abnormal amount and in individual cases it may even exceed 80 mL. A small volume of SF, however, is not a criterion for excluding an intra-articular disorder.

Obtaining a SF sample can be more difficult in the presence of various particles (fibrin, inclusion bodies) resulting in a falsely low SF volume.

In general, the more a joint is inflamed the greater the fluid volume.

49.3.2 Inspection

Normal SF, and material found in non inflammatory effusions, is of a lightly yellowish tint to straw color and looks transparent. In inflammatory joint diseases the color commonly shifts to turbid: the higher the cell count the higher the turbidity. A more greenish, grayish, xanthochromic to creamy milky grayish appearance is seen in pyogenic arthritis.

A reddish tinge to the SF is caused by the accumulation of erythrocytes which have migrated from the capillaries into the joint space. Large numbers of erythrocytes give the appearance of hemarthrosis /4/.

Hemarthrosis and hemorrhagic SF

Hemarthrosis means bleeding into the joint cavity. Hemmorhagic SF is artificial hemorrhagic SF (e.g. due to blood vessel damage during the arthrocentesis). Hemorrhagic SF is relatively easy recognizable by the fact that there are streaks of blood.

Refer to Tab. 49-3 – Disorders causing hemarthros and hemorrhagic SF.

49.3.3 Viscosity

The higher the cell count and the turbidity of the SF, the lower the viscosity. The SF viscosity is mainly dependent on the content of highly polymerized hyaluronate (HY) /8/. The viscosity is not only dependent on the HY concentration but also on the temperature, the protein content, the type of proteins as, alteration of the HY-protein complex, cells and enzymes. The decline in viscosity associated with inflammatory processes is a useful diagnostic criterion.

Determination of viscosity

As part of the “string test” a drop of SF is placed between thumb and index finger. By gradually spreading apart the fingers a string is formed which, assuming normal HY, reaches a length of more than 3 cm before it breaks. SF of the inflammatory or septic type produces much shorter or no strings at all. Because of the infection risk the test should not now be carried out as described, especially since other techniques provide identical results.

After obtaining a SF sample using a syringe the needle is removed and the SF dropped into a small tube. The length of the drop provides an indication regarding the viscosity. Low viscosity results in a short tail.

A few drops of the SF are placed in a small glass dish and a loop is dipped in. Pulling out the loop will result in a string the length of which provides information about the SF viscosity.

Exact measurements can be performed using a viscometer.

49.3.3.1 Mucin clot test

According to reference /3/ this test is not necessary, but it is commonly performed because of its simplicity.

Mucin is a highly polymerized complex composed of hyaluronate and protein and it is responsible for the high viscosity of SF /3/. During joint inflammation fragmentation of mucin occurs. The test result correlates with the viscosity measurement and the leukocyte count.

Determination

According to the original protocol 4 parts of 2% acetic acid are added to 1 part of SF and gently shaken.

Variant: 2 to 3 mL of 1% acetic acid are placed in a tube, 2 drops of SF are added and gently shaken. The resultant precipitate is classified into three categories by its appearance which depends on the degree of HY polymerization:

  • Good mucin precipitation (solid clot); normal, arthritic, or traumatic SF
  • Moderate mucin precipitation (several particles, fragmented appearance); indicative of inflammatory activity
  • Poor mucin precipitation (white flaky precipitate, snow flakes); highly active synovial inflammation.

49.3.3.2 pH value

During the course of joint inflammation the H+ ion concentration in the SF changes. Therefore pH measurement is a useful marker for the local inflammatory activity /9/. The H+ ion concentration correlates with markers of local inflammation, e.g. the PMN count or the acid phosphatase activity /9/.

The pH of normal SF is 7.31–7.64 (mean 7.43) /10/. In cases of osteoarthritis the value ranges from 7.25 to 7.54 (mean 7.38) /9/; other authors /11/ indicate a range of 7.5 to 8.0.

The pH in the presence of inflammation declines on average to 7.22 /10/. Other authors /9/ indicate a range of 6.85 to 7.41 (mean 7.19) for rheumatic joint diseases. According to reference /11/ a decrease below 7.5 is suggestive of an inflammatory process.

In cases of bacterial infection due to the associated increasing lactate concentrations a low pH is found. However, investigations /9/ have shown that even in rheumatoid arthritis pH values of < 7.0 can be found without the presence of sepsis. A low pH in the SF is thus a good marker for inflammation but not for sepsis.

Determination

A semi quantitative measurement using a special indicator strip is sufficient; or an acid/base analyzer can be used for measuring the H+ concentration /9/.

49.3.3.3 Cell count

Two types of conditions affect joints:

  • Non inflammatory, the cell count is low (most commonly osteoarthritis)
  • Inflammatory, the cell count is increased and differential cell count provides differential diagnostic information (inflammatory synovitis).

The determination of the cell count is an important part of SF analysis because the lack or presence of leukocytes (number/μL SF) is diagnostically useful /4/. The leukocyte count has a higher diagnostic relevance if the SF is repeatedly sampled at various time intervals and the cell count determined each time.

Determination

The counting is best performed by using a cell counting chamber. The use of automated cell counters is problematic and sometimes provides false results because extracellular components may clog the instrument. Normally 150 mmol/L NaCl is used for diluting. In the case of SF which may contain erythrocytes hypotonic (50 mmol/L) NaCl is used to lyse them.

Clinical significance

Intervals of the leukocyte count show that there is no strict delineation between leukocyte counts in the different disease categories.

Refer to

Even in normal joints the limit of the leukocyte count reference interval is variable. 50–100 leukocytes/μL SF are considered to be normal /12/. Leukocyte counts of 200–1,000/μL are found in non-inflammatory SF although such values can occur in mildly inflamed joints. Exceptions are seen in patients with predominantly degenerative diseases. In hemochromatosis inflammatory effusions are mimicked by the presence of high leukocyte counts if an associated chondrocalcinosis has caused crystal-induced arthritis. Leukocyte counts > 60,000/μL always lead to the suspicion of underlying infection. Infections under treatment may, however, show lower leukocyte counts. In the case of rheumatic diseases, psoriatic arthritis, Reiter’s syndrome, or crystal-induced arthritis the cell count can reach 350,000/μL /12/.

Leukocyte count in SF obtained from finger joints commonly shows very high values due to the low volume.

Possible error sources

Because of mucin precipitation, diluents containing acetic acid should not be used in conjunction with mechanized cell counting equipment since the precipitate also contains leukocytes resulting in falsely low cell count.

49.3.3.4 Differential cell count

Determination

For the differential cell count SF samples with leukocyte counts > 6,000/μL are used for the smear, SF samples low in cell count are concentrated by centrifugation (10 min. at 1,000–3,000 rpm). After removing the supernatant the sediment is reconstituted by adding a few drops of the supernatant. To avoid cellular morphological artifacts the preparation of the microscope slide should be conducted soon after obtaining the SF sample. The SF viscosity sometimes renders the preparation of thin smears difficult. Thick smears interfere with the assessment due to stain uptake by extracellular components. The air dried smears are stained with eosin-methylene blue according to May-Grünwald /13/ or Giemsa-Wright /3/. The Wright stain is also used for the detection of lupus erythematosus cells in SF /6/.

Clinical significance

In normal SF the mean values found are 24% lymphocytes and 48% monocytes; the proportion of PMN leukocytes is approximately 7% /14/. The SF cell composition changes at the onset of inflammation. The following cells can be observed: PMN, small lymphocytes, monocytes, eosinophils and erythrocytes. In addition, synovial lining cells, macrophages, LE cells, stimulated mononuclear cells, plasma cells /12/.

The proportion of PMN leukocytes in SF broadly equates the degree of inflammation. The percentage increases in SF of the non-specific inflammatory type on average to greater than 70% and in SF associated with septic arthritides to greater than 95% /12/. The PMN leukocyte percentage also depends on the stage of the disease: during the early stages of rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), rheumatic fever, scleroderma, and various infections lower values are found.

Effusions containing only mononuclear cells are found in early RA and in some viral arthritides.

A predominance of eosinophilic cells may be seen in parasitic arthritides, hyper eosinophilic syndrome and other hyper eosinophilic conditions /4/.

In general, × 400 magnification is adequate. LE cells are rare in seropositive RA-SF (3%), found in 14% of Reiter’s syndrome cases, and frequently seen in SLE (45%) /12/. The occurrence of LE cells in SF has been described in cases of medication induced lupus erythematosus /6/.

Ragocytes

Ragocytes represent PMN leukocytes or monocytes in SF that contain pale green to olive green inclusion bodies within the cytoplasm. These inclusion bodies are composed of immunoglobulin, rheumatoid factors, fibrin, and antinuclear factors and their size varies between 0.20 and 0.33 μm /12/. Ragocytes or RA cells (rheumatoid arthritis cells) are found in seropositive RA (20–90%) and also in cases of septic arthritis and acute crystal induced arthritis /1215/. If the latter two diseases have been ruled out a finding of > 60% ragocytes in the SF indicates that the patient has, had, or may develop seropositive RA /12/.

Possible error sources

To avoid cellular alterations the preparation of the smear should be done as soon as possible; staining can take place later.

49.3.3.5 Analysis of crystals

The detection of native SF crystals, besides the cell count determination, is one of the most important parts of the entire SF analysis. The finding of sodium urate and calcium pyrophosphate crystals is one of the few pathognomonic tests in the field of rheumatology. The examination for crystals can also be conducted in patients from whom only one drop of SF can be aspirated from a joint (e.g., the finger joint) /3/. Sodium urate and calcium pyrophosphate crystals can be identified from joints during acute attacks of gout or pseudo-gout. They may also be identified from more chronically inflamed joints and in SF aspirated from an inter critical, asymptomatic and clinically non inflamed joint /4/.

Microscopy of crystals

Sodium heparin or EDTA are used as anticoagulants for the SF sample. A drop of well mixed SF is placed onto the slide with a cover slip. The slide is then sealed with shellac or clear nail varnish. To avoid artifacts the SF should be prepared as soon as possible after joint aspiration.

The initial crystal detection step is carried out first under light microscope, to identify crystals by their geometric, shape line morphology (this shows calcium pyrophosphate crystals well), then under plain polarized light to identify crystals by birefringence (this readily detects sodium urate crystals) /4/. All urate crystals are strongly birefringent and are clearly distinguished by means of compensated polarized light microscopy. Calcium pyrophosphate crystals are non birefringent or only weakly birefringent. In a study /16/ only 20% of calcium pyrophosphate crystals showed sufficient birefringence to be identified as birefringent. Thus, using compensated polarized light microscopy alone, only one in five calcium pyrophosphate crystals showed sufficient birefringence /4/.

Sodium urate crystals

These crystals are usually needle-shaped, 10–20 μm long but may be short and round-edged. In polarized light the crystals are strongly birefringent and are bright yellow. They are usually found extracellularly although during acute disease increased phagocytosis by PMN leukocytes may occur. In that case the crystals, unlike calcium pyrophosphate crystals, extend beyond the cell margin. The detection of SF sodium urate crystals confirms the diagnosis of gout.

Calcium pyrophosphate crystals

Calcium pyrophosphate crystals (Ca2P2O7 × 2H2O) are rod shaped, rhomboid, and prismatic /4/. They are weakly birefringent when parallel to the axis of the compensator and bright blue when perpendicular to it /4/. Calcium pyrophosphate crystals are not as numerous as urate crystals and phagocytized crystals do not extend beyond the cell border. Another criterion for their differentiation from urate is their solubility in 10% EDTA solution. Their presence in SF is considered to confirm underlying chondrocalcinosis (pseudo-gout).

Hydroxy apatite crystals /4/

The crystals are too small to be seen by light microscopy, but the use of calcium stains permits visualization. In the phase contrast microscope the crystals (Ca5(PO4)3OH) appear inside PMN or extracellularly as glowing disks with a diameter of 3–65 μm.

Calcium oxalate and lithium heparin crystals

Both substances can form crystals in SF when used as anticoagulants; therefore they should not be used for this purpose. Calcium oxalate crystals usually have a cuboid shape (Maltese crosses), lithium heparin crystals are pleomorphic with a size of 2–5 μm. Both crystal types can be phagocytized by PMN. Calcium oxalate crystals are identified in SF from joints of patients undergoing periodic hemodialysis and also in those with hyperoxalemia and oxalosis /4/.

Cholesterol crystals

The crystals appear as large rhomboidal plate, often with a broken or folded corner and showing bright, mixed birefringence. They are found in chronic largely inflammatory effusions /4/.

Amyloid

Sometimes fragments of synovial lining cells containing amyloid are found in the SF. It is detected by Congo red staining /3/ and by green birefringence in polarized light. Amyloid detection in the SF is considered to be a sensitive marker for the presence of amyloidosis associated arthropathy /17/.

Refer to Tab. 49-5 –Classification of joint effusions.

49.4 Microbiological investigations

Some authors consider a bacteriological SF examination only worthwhile if the leukocyte count is above 60,000/μL /2/. However, usually infections caused by gonococci, mycobacteria, or fungi show lower leukocyte counts. Positive bacteriological findings are normally only associated with pyoarthritis.

Specimen

Injection of SF in blood culture bottles is the gold standard for the diagnosis of septic arthritis. Standard blood culture media are used and selective media for gonorrhea, mycoplasma, and tuberculosis /19/. Molecular biological tests are used for the diagnosis of M. tuberculosis and N. gonorrhea. For Gram staining SF sediment is used; for the culture bottles 10–15 mL of SF without anticoagulant is used.

Recommended bacteriological examinations

Gram stain: to differentiate Gram positive and Gram negative bacteria. Gram stain has a diagnostic sensitivity and specificity of 37% and 99%, respectively /20/. Negative Gram staining does not rule out septic arthritis. The assessment is rendered more difficult by mucin artifacts. The Gram staining of a microscopic smear should be the initial step in any SF analysis because appropriate antibiotic therapy may be implemented /21/.

Ziehl-Neelsen stain: detection of acid-resistant rods in suspected tuberculous arthritis (rare). The differentiation is better accomplished using the auramin-fuchsin stain in conjunction with a fluorescence microscope /22/.

Cultures: bacteria of positive culture bottles are cultivated aerobic, anaerobic, and aerobic with CO2 /22/. Heated blood agar (chocolate agar) is used as the universal medium (e.g., for N. gonorrhea and H. influenzae).

49.4.1 Microbiological diagnosis of septic arthritis

Microbiological investigations of SF are the key to the confirmation of infectious conditions. Septic arthritis is a major complication in both immunocompetent and immunocompromised hosts /18/. The hematogenous route of infection is the most common route and is usually associated with one pathogen. Septic arthritis depends on the etiology and may present as acute mono-articular arthritis, chronic mono-articular arthritis, polyarticular arthritis or prosthetic joint infection /18/. Predisposing factors are age greater than 80 years, diabetes mellitus, rheumatoid arthritis, prosthetic joint, recent joint surgery, skin infection and cutaneous ulcers, intravenous drug abuse and alcoholism, and previous intraarticular corticosteroid administration.

The most common causative organisms in non gonococcal septic arthritis are Staphylococcus aureus, especially methicillin resistant S. aureus (MRSA), Streptococcus sp., Klebsiella pneumoniae (in Asia) and other Enterobacteriaceae, Pseudomonas aeruginosa and Burkholderia pseudomallei (endemic countries) /18/.

49.5 Biochemical investigations

The following biochemical tests are of clinical relevance: total protein, glucose, lactate, uric acid, enzymes.

SF is considered to be the dialysate of plasma. Therefore almost all components found in SF are also contained in plasma. Frequently there is a quantitative difference between serum and SF with SF levels commonly being lower. Higher concentration of substances in SF than in the serum are suggestive of local intraarticular synthesis.

Preparation

The biochemical examinations usually require pretreatment with hyaluronidase to reduce the viscosity of SF. For this purpose sheep testicular hyaluronidase can be used (25 mg hyaluronidase are incubated with 1 mL SF at 37 °C for 5 min).

Total protein

In the healthy joint the synovial membrane is impermeable to high molecular weight proteins. Its permeability increases with progressive inflammation /23/ resulting in higher molecular weight proteins (e.g., fibrinogen) passing through the synovial membrane. The lack of fibrinogen in normal SF is also the reason why no coagulate can form within it. The formation of coagulates only occurs with the onset of inflammatory processes and an increase in synovial membrane permeability.

The determination of total protein in SF is performed by using the Biuret reaction. The mean value for the total protein concentration in SF in healthy joints is approximately 13 g/L /24/, a range of 11–22 g/L has been published /2/. The mean value in non inflammatory SF is approximately 32 g/L and increases to 44 g/L in SF associated with rheumatoid arthritis /25/. Lower values are always measured in the SF than in serum taken at the same time /25/.

Protein fractions

Electrophoresis shows a proportional increase in the γ-globulin fraction with a concurrent decrease in albumin in SF of the inflammatory type /2627/.

High molecular weight proteins, such as α2-macroglobulin present in only small amounts in normal SF, are moderately elevated in SF from inflamed joints.

Glucose

The normal SF glucose concentration is approximately the same or slightly lower than in blood. The difference between blood and SF values is approximately 10 mg/dL (0.5 mmol/L). In the case of joint inflammation the SF glucose may decline to 40 mg/dL (2.2 mmol/L) lower than in the blood /3/. A decrease in the SF glucose concentration down to 20 mg/dL (1.1 mmol/L) is suggestive of an infectious arthritis. The glucose concentration helps to differentiate between inflammatory and infectious arthritides /3/, however there is a significant overlap.

Possible sources for error: attention must be paid to the fact that:

  • For a valid assessment of the SF glucose, a simultaneous serum glucose determination is necessary with the patient fasting for at least 8 h prior to sample collection
  • SF must be collected in sodium fluoride containing tubes otherwise the glucose concentration will be lowered by continuing glucose metabolism of the SF leukocytes resulting in falsely low values.

Lactate

The lactate concentration, like glucose, is an indicator of local inflammation. In contrast to glucose the lactate level increases with the activity of the inflammation. This is mainly due to the increased glycolysis of leukocytes which, in an inflammatory joint effusion, are present in very large numbers. The pH of SF declines simultaneously.

Occasionally it may be difficult to diagnose joint infections by microscopy if the SF is not purulent or the Gram stain negative. In such cases lactate determination can rapidly give information about the possible infection. Diagnosis by bacteriologic cultures may take too long because rapid cartilage destruction may occur without treatment.

According to reference /28/ the SF lactate concentration varies in each of the major disease categories. There is no correlation between the lactate value and other tests such as the leukocyte count, C3, C4, total protein, or glucose.

Differentiation between acute mono-articular arthritis, septic arthritis and other inflammatory arthritides based on the lactate concentration is not yet possible /28/.

Uric acid

The more superficial capillaries close to the lining cells of the articular cartilage are fenestrated facilitating rapid change of the uric acid; the concentration is identical to that in the blood. In the case of uric acid induced (gouty) arthritis the solubility limit in SF is the same as in the serum at 6.4 mg/dL (381 μmol/L). The gold standard for a diagnosis of gout relies on the demonstration of monosodium urate crystals in fluid or tophus aspirates, as it has 100% diagnostic specificity. Crystals can be detected by polarized light microscopy in SF aspirated from the first metatarsophalangeal joint from both symptomatic and asymptomatic joints. This means that a diagnosis of gout can be established even during the asymptomatic inter critical period after, or between gout flares, so called inter critical gout /29/.

Enzymes

The enzyme activities in SF are qualitatively similar to those in plasma. Enzymes belonging to the known six groups of enzymes (oxidoreductases, transferases, hydrolases, lyases, isomerases, and ligases) have been detected in SF. The origin for these enzymes is from the plasma, synovial cells, rheumatic granulation tissue, leukocytes, and possibly erythrocytes. The largest proportion is constituted of lysosomal enzymes originating mainly from the granules of PMN leukocytes. Therefore SF rich in cells always contains higher enzyme activities than SF with a low cell count, e.g., as in degenerative joint diseases; accordingly the results of individual enzyme determinations can be used for the differential diagnosis of non inflammatory vs. inflammatory SF. A differential diagnosis of inflammatory joint diseases is, however, not possible /30/.

Normally SF enzyme activities are always lower than those in plasma. Only very few enzymes have a diagnostic relevance: lactate dehydrogenase (LD), acid phosphatase (ACP), alkaline phosphatase (ALP), and aldolase. The latter, as is the case with all glycolytic enzymes, may be elevated in the SF in parallel to the activity of the underlying inflammation but the analysis is of no additional value. The same applies to the lysosomal enzymes /30/.

LD: the activity is strongly elevated in inflammatory joint effusions with the M-isoenzyme especially increased /31/.

ACP and ALP: both enzyme activities originate from blood and cartilage cells.

ACP in the SF of arthritic patients is on average significantly lower than, for example, in patients with rheumatoid arthritis (RA) /3132/.

In the case of ALP local synthesis in synovial tissue is presumed. There is no correlation between the leukocyte count, as a marker for the inflammatory activity, and the enzyme activity /33/. Acute traumatic meniscous and capsule-ligamentous injuries are associated with a greatly elevated ALP /34/. SF of patients with RA, as well as those with psoriatic arthritis, shows, significantly higher ALP activity than that associated with degenerative joint diseases. In RA there is no difference between serum and SF-ALP /33/.

For ACP determination heparin or oxalate should not be used as the anticoagulant because they both inhibit the enzyme activity.

Refer to Tab. 49-6 – Synovial fluid findings in joint diseases.

49.6 Immunological and inflammatory markers of the synovial fluid

The determination of immunological and inflammatory markers in the SF is of little diagnostic value but may provide some better differential diagnostic value.

Rheumatoid factors

Occasionally rheumatoid factor is not present in the blood but may be detected in the SF /35/. For this reason the determination of the rheumatoid factor is diagnostically relevant only if is negative in serum (seronegative RA).

Antinuclear factors

Antinuclear factors are found in SF associated with various rheumatic diseases when they are not detectable in serum; their clinical significance has not been reliably determined yet and therefore they are diagnostically not relevant.

Immunoglobulins

SF immunoglobulins are synthesized in the synovial membrane. The SF immunoglobulin concentration is usually lower than that in serum. Degenerative joint diseases on average show lower immunoglobulin levels than joint inflammation in which a marked increase in IgG and IgM is seen. In the case of RA the IgE concentration is significantly elevated in both SF and serum /25/.

Complement

Complement concentrations of SF are only relevant in comparison with the complement levels of serum /36/. Sometimes the albumin concentration is used as reference factor for correction /37/.

Total hemolytic complement CH50 activity

The measurement is of limited value for detecting and diagnosing joint diseases /38/. In comparison to serum the CH50 of SF is decreased in approximately 60% of patients with RA and crystal-induced arthritides /39/.

There is no relationship of CH50 activity to the inflammatory activity of the joint, the leukocyte count, the protein concentration /38/ or the IgM rheumatoid factor /39/.

The CH50 is commonly low in the SF of patients with RA in comparison to other types of exudative SF. Patients with severe seropositive diseases also have low CH50 values /40/.

Complement C3 and C4

Decreased SF complement levels are commonly present in RA and SLE; in cases of probable RA, gout, chondrocalcinosis, and septic arthritis they may be low but this is rare in psoriatic arthritis /41/. The presumed reason for the complement reduction in gout and chondrocalcinosis is that the crystals activate the complement cascade by binding IgG. In plasma of patients with RA the concentration of C3 and C4 is usually normal /42/.

Possible error sources

  • The SF was not immediately centrifuged following arthrocentesis
  • The analysis was not performed immediately, instead the samples were stored
  • SF storage was not carried out at –70 °C.

Cytokines

IL-1 and TNF-α are the most important pro inflammatory cytokines in the SF and are synthesized by monocytes/macrophages in response to cell destruction in the joint /43/. They stimulate the synthesis of metalloproteinases by various cells, are responsible for the increased synthesis of mediators of inflammation (PGE2), and thus facilitate collagen and proteoglycan metabolism in the joint cartilage. The cytokine concentrations measured in inflammatory joint diseases are variable probably because of the presence of SF inhibitors interfering with some immunoassays. Concentrations of IL-1, TNF-α, IL-6, IL-8 and the soluble cytokine receptors sIL-2R and sTNF-R are on average higher in RA than in osteoarthritis. This may be attributable, for example, to the larger mass of hyperplastic synovial tissue present in RA. IL-1 and TNF-α are also potent stimulators of IL-6, the main mediator of the acute phase response in RA. The IL-6 concentration in patients with RA is significantly higher in SF than in plasma because of local synthesis in synovial tissue. The role of IL-8 in SF appears to be the activation and chemoattraction of PMN. IL-8 correlates with the SF PMN count.

There is probably a relationship between disease activity (e.g., as seen in RA) and the concentration of various cytokines and cytokine receptors in the SF but there is no unanimous agreement.

Clinical significance: for clinical diagnosis the determination of SF cytokines is not necessary as the classification of the SF into disease categories (inflammatory vs. noninflammatory) can be done more easily using other tests.

Inflammation markers

Matrix metalloprotease-3 (MMP-3), soluble vascular adhesion molecule 1 (sVCAM-1), soluble intercellular adhesion molecule (sICAM-1), vascular endothelial growth factor (VEGF), tissue inhibitor of metalloproteases 1 (TIMP-1) and matrix metalloprotease 1 (MCP-1) were all associated with SF inflammation by etarfolatide imaging, radiographic osteoarthritis severity and/or osteoarthritis symptoms in individuals with an inflammatory osteoarthritis endotype /44/.

Alpha defensin plus CRP seemed to be the most helpful combination for pre-operative discrimination of patients undergoing revision hip and knee arthroplasty on the basis of aseptic implant failure, acute high-grade infection, or (suspected) low-grade infection /45/.

49.7 Clinical significance

The clinical relevance of SF findings is based on the differential diagnostic classification of the 4 major disease categories as outlined in Tab. 49-1 – Diseases with joint effusions and their classification into four major categories. Joint effusions can be classified into non-inflammatory, inflammatory, septic and traumatic. For laboratory findings and differential diagnostic criteria refer to:

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9. Farr M, Garvey K, Bold AM, Kendall MJ, Bacon FA. Significance of the hydrogen ion concentration in synovial fluid in rheumatoid arthritis. Clin Exp Rheumatol 1985; 3: 99–104.

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11. Pfister SA, Hauke G, Peter HH. Synovial analysis. Akt Rheumatol 1989; 14: 51–7.

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15. Hollander JL, McCarty DJ, Rawson AJ. The “RA cell”, “ragocyte”, or “inclusion body cell”. Bull Rheum Dis 1965; 16: 382–3.

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17. Lakhanpal S, Li CY, Gertz MA, Kyle RA, Hunder GG. Synovial fluid analysis for diagnosis of amyloid arthropathy. Arthritis Rheum 1987; 30: 419–23.

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19. Von Essen R, Hölttä A. Improved method of isolating bacteria from joint fluids by the use of blood culture bottles. Ann Rheum Dis 1986; 45: 454–7.

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22. Freeman R, Jones MR. Microbiology. In: Jeffery MS, Dick WC, eds. The role of the laboratory in rheumatology. Clin Rheum Dis, vol 9. London: Saunders, 1983: 3–26.

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25. Hunder GG, Gleich GJ. Immunoglobulin E (lgE) levels in serum and synovial fluid in rheumatoid arthritis. Arthritis Rheum 1974; 17: 955–63.

26. Decker B, McKenzie BF, McGuckin WF, Slocumb CH. Comparative distribution of proteins and glycoproteins of serum and synovial fluid. Arthritis Rheum 1959; 2: 162–77.

27. Binette JP, Schmid K. The proteins of synovial fluid: A study of the alpha1/alpha2 globulin ratio. Arthritis Rheum 1965; 8: 14–28.

28. Arthur RE, Stern M, Galeazzi M, Baldassare AR, Weiss TD, Rogers JR, Zuckner J. Synovial fluid lactic acid in septic and nonseptic arthritis. Arthritis Rheum 1983; 26: 1499–1505.

29. Richette P, Doherty M, Pascual E, Barskova V, Becec F, Castaneda J, et al. 2018 updated European League against Rheumatism evidence-based recommendations for the diagnosis of gout. Ann Rhum Dis 2019; 0: 1–8.

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31. Müller W. Examination of the synovial fluid. Laboratoriumsblätter 1976; 26: 25–35.

32. Lehman MA, Kream J, Brogna D. Acid and alkaline phosphatase activity in the serum and synovial fluid of patients with arthritis. J Bone Joint Surg 1964; 46-A: 1732–8.

33. Cimmino MA, Dato G, Cutolo M. Synovial fluid alkaline phosphatase. Arthritis Rheum 1987; 30: 235–7.

34. Dingerkus MD, Jochum M, Fritz H, Bernett P. Possibilities for biochemical differentiation of irritant effusions involving the knee joint. Sport injury – Sport damage 1987; 2: 86–90.

35. Rodnan GP, Eisenbeis CH, Creighton AS. The occurrence of rheumatoid factor in synovial fluid. Amer J Med 1963; 35: 182–8.

36. Bunch TW, Hunder GG, McDuffie FC, O’Brien PC, Markowitz H. Synovial fluid complement determination as a diagnostic aid in inflammatory joint disease. Mayo Clin Proc 1974; 49: 715–20.

37. Mollnes TE, Lea T, Mellbye OJ, Pahle J, Grand O, Harboe M. Complement activation in rheumatoid arthritis evaluated by C3d and the terminal complement complex. Arthritis Rheum 1986; 29: 715–21.

38. Sheppeard H, Lea DJ, Ward DJ. Synovial fluid total hemolytic complement activity in rheumatic diseases – a reappraisal. J Rheumatol 1981; 8: 390–7.

39. Kim HJ, McCarty DJ, Kozin F, Koethe S. Clinical significance of synovial fluid total hemolytic complement activity. J Rheumatol 1980; 7: 143–52.

40. George D, Glass D. Quantitation of complement proteins in rheumatic disease. In: Jeffery MS, Dick WC, eds. The role of the laboratory in rheumatology. Clin Rheum Dis 9. London: Saunders, 1983: 177–98.

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45. Ettinger M, Savov P, Calliess T, Windhagen H, Lichtinghagen R, Lukasz A, Omar M. Improved diagnostic accuracy with the classification tree method for diagnosing low.grade periprosthetic joint infections ba quantitative measurement of synovial fluid alpha-defensin and C-reactive protein. International Orthopaedics 2019; https://doi.org/10.1007/s00264-019-04338-6.

46. Nade S. Septic arthritis. Balliere’s Clinical Rheumatology 2003; 17: 183–200.

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48. Gaston JSH, Lillicrap MS. Arthritis associated with enteric infection. Balliere’s Clinical Rheumatology 2003; 17: 219–39.

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51. Bardin T. Gonococcal arthritis. Balliere’s Clinical Rheumatology 2003; 17: 201–8.

52. Liebling MR, Arkfeld DG, Michelini GA Nishio MJ, Eng BJ, Jin T, et al. Identification of Neisseria gonorrhoeae in synovial fluid using the polymerase chain reaction. Arthritis Rheum 1994; 37: 702–9.

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54. Paish HL, Baldock TE, Gillespie CS, Del Caprio Pons A, Mann DA, Deehan DJ, et al. Chronic active inflammation in patients with failed total knee replacements undergoing revision surgery. J Orthop Res 2019; 37: 2316–24.

55. Ettinger M, Savov P, Callies T, Windhagen H, Lichtinghagen R, Lukasz A, Omar M. Improved diagnostic accuracy with the classification tree method for diagnosing low-grade periprosthetic joint infections by quantitative measurement os synovial fluid alpha-defensin and C-reative protein. International Orthopaedics 2020; 44 (1): 31–8.

56. Judkins SW, Cornbleet PJ. Synovial fluid analysis. Laboratory Medicine 1997;28: 773–9.

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Table 49-1 Diseases with joint effusions and their classification into four major categories /356/

1. Non inflammatory effusions

Osteoarthritis

Traumatic joint disease

Avascular necrosis

Osteochondrosis dissicans

Osteochondroma

Rheumatic fever*

Myopathy associated with
hypothyroidism (myxedema)

Acromegaly

Arthropathy associated with
hemochromatosis

Gaucher’s disease

Arthropathy with ochronosis

Paget’s disease

Arthropathy associated
with sickle cell anemia

2. Inflammatory effusions

Rheumatoid arthritis

Crystal induced synovitis

  • Uric acid related arthropathy (gout)
  • Chondrocalcinosis (pseudo-gout)
  • Hydroxyapatite
  • Corticosteroids

Psoriatic arthritis

Reactive arthritides

  • Reiter’s syndrome
  • Enteropathic arthritis
  • Ulcerative colitis
  • Arthritis following bacterial

infections (e.g., Yersinia)

Whipple’s disease

Systemic lupus erythematosus* (SLE)

Skleroderma

Polyarteritis

Scleroderma*

Polymyositis*

Vasculitis

Charcot’s arthopathy

Villonodular arthritis
(benign synovioma)

Medication induced
systemic lupus erythematosus

Arthropathy associated with
amyloidosis

Polymyalgia rheumatica

Polychondritis

Sarcoidosis

Behçet’s syndrome

Ankylosing spondylitis

Juvenile rheumatoid arthritis

Rheumatic fever*

Agammaglobulinemia

Infective arthritis

  • Viruses
  • Bacteria
  • Mycobacteria

Hypersensitivity

– Arthritis associated with
serum sickness

3. Septic effusions

Bacterial infections

4. Hemorrhagic effusions

Trauma related

Charcot’s arthropathy

Hemorrhagic diathesis

  • Therapy with anticoagulants
  • Hemophilia

Hemangioma

Tumors

  • Villonodular arthritis
  • Synovial hemangioma

* Can belong to either group 1 or 2

Table 49-2 Examinations for synovial fluid analysis

Basic examinations

  • Volume
  • Color
  • Clarity
  • Viscosity
  • Mucin
  • pH

Biochemical tests

  • Protein
  • Glucose
  • Lactate
  • Uric acid

Hematological examinations

  • Cell count
  • Differential cell count

Immunological tests

  • Rheumatoid factor
  • Immunoglobulins
  • Complement

Analysis of crystals

  • Uric acid
  • Calcium pyrophosphate

Microbiology

  • Gram stain
  • Culture
  • PCR

Table 49-3 Disorders with hemarthrosis and hemorrhagic synovial fluid (SF) /1/

Predominant

Hemarthrosis

Hemorrhagic SF

Trauma

  • With fracture
  • Without fracture

Hereditary hemorrhagic disease

  • Classical hemophilia
  • Coagulation disorder

Therapy with anticoagulants

Tumors

  • Malignant synovioma
  • Hemangioma

Osteoarthritis

Chondrocalcinosis

Neuropathic arthropathy

  • Charcot joint

Pyogenic arthritis

Chronic arthritis

Sickle cell anemia

Tumor

  • Benign synovioma
    (villonodular, pigmented synovitis)

Tab. 49-4 PMN counts in dependence of the proportion (%) of patients with joint disease

Count (109/l)

0–0,2

0,2–2,5

2,5–25

25–50

50–100

> 100

Rheumatoid arthritis

6 (26)

67 (69)

23 (86)

4 (88)

Gout

3,5

21 (38)

44 (73)

19 (91)

10 (91)

2,5 (92)

Chondrocalcinosis

3

29 (35)

45 (80)

13 (86)

10 (92)

Bacterial arthritis

12 (94)

18 (88)

30 (94)

40 (95)

* Number of patients with joint disease are shown in brackets

Tab. 49-5 Classification of joint effusions 

Investi-
gation

Normal

Non-
inflam-
matory

Inflam-
matory

Septic

Traumatic

Aspect

Straw-
yellow,
Clear

Bright-
straw

Straw-
yellow,
Clear

Green

Grey-
yellow,
Increasing
turbid

Grey-
yellow,
Turbid-
purulent

Yellow-
brown,
Bloody,
Clear-
turbid

Viscosity

High

Normal/
reduced

Reduced

Variable

Normal/
reduced

Leukocyte
count (109/L)

< 0.2

0.2–2.0

3.0–50.0

≥ 50

≤ 2.0

PMN (%)

10–25

≤ 25

often > 50

> 85

25

Bacteria

Neg.

Neg.

Neg.

Often
positive

Neg.

Crystals

Neg.

Neg.

Possible

Negative

Neg.

Neg., negative

Tabelle 49-6 Synovial fluid findings in joint diseases

Disease

Type

Aspect

Vis-
cosity

Leukocytes
(109/L)

PMN
(%)

Arthrosis

R

Amber-yellow, Clear

N

0,05–2,0

< 25

Chronic
polyarthrosis

I

Yellow-green,
Turbid, Beflocking

R

0,25–80

70–90

Bechterew’s
disease

I

Yellow, Clear
Slightly cloudy

R

1,0–10

≈ 50

Reiter’s
disease

I

Yellow-green,
Turbid

R

0,1–50

≈ 60*

SLE

I

Yellow, Clear
Cloudy

Rs

0–10

≈ 25

Arthritis urica

I

Yellow-milky, turbid

R

0,1–160

> 75

Chondro-
calcinosis

I

Yellow-milky
Turbid

R

0,05–75,
often > 20

> 50

Acute bacterial
Arthritis

I

Gray-creamy,
Strongly turbid

Low

15–230

95

Trauma

R

Yellow,
Perhaps bloody,
Clear or turbid

N to
high

0,05–6.5

≈ 20

Tuberculosis

I

Gray and yellow,
Turbid- beflocking

R

20–150

50–60

R, reactive; Rs, slightly reactive; I, inflammatory; N, normal; * later lymphocytic

Table 49-7 Data of normal and pathological synovial fluid (SF)

Examination

Normal SF

Non-
inflammatory
effusion

Inflam-
matory
effusion

Septic effusion

Traumatic
effusion

Volume (mL)

Up to 3.5

> 3.5

Up to 80

> 3.5

> 3.5

Appearance

Straw-yellow
clear

Light straw-
yellow, clear

Green-
grayish,
yellow1)

Grayish yellow,
turbid-purulent

Creamy
yellow,
clear, turbid,
sanguineous

Viscosity

> 3 cm string

> 3 cm string

< 3 cm string

< 3 cm string

> 3 cm string

Mucin

Good

Good

Moderate/
poor

Poor /44/

Good

pH

7.31–7.64 /10/

7.25–7.54 /9/

6.85–7.41 /9/

  • Mean value

7.43

7.38

7.19

Leukocytes
(μL)

< 200

< 2,000

6,000–
40,000

Up to 200,000

< 2,000

Lymphocytes/
monocytes

Approximately
90%

PMN (%)

< 25 

< 25

77

80–95 /2844/

25

Bacteria

Negative

Negative

Negative

Positive

Negative

Total protein
(g/L)

11–22

Normal value

> 40

30–60 /44/

20–30

Uric acid
(μmol/L)

178–416

Normal value

Normal value

Normal value

Normal value

Glucose
(mmol/L)

3.3–5.3

Normal value

Decreased4)

1.1–1.7 /29/

Normal value

Lactate
(mmol/L)

1.0–1.8

Up to 4.2 /29/

Up to 6.9 /30/

See 2)

Normal value3)

Crystals

Negative

Negative

Possibly

Negative

Negative

LD (U/L)

< 200

Normal value

> 200

> 300

< 200

Acid phosphatase (U/L)

2.3 /32/

6.7 /32/

Increased

Normal value

Rheumatoid
factor

Negative

Negative

Positive/
negative

Negative

Negative

Immuno-
globulins

Approx.1/2 of
plasma value

Approx. 1/2 of
plasma value

Increased

Increased

Plasma value

1) increasing turbidity is activity-dependent

2) gonococcal infection up to 5.6 mmol/L /29/

3) normal lactate value in the case of traumatic effusions is suggestive of artificial blood contamination

4) in conjunction with leukocyte count > 20,000/μL

Table 49-8 Laboratory findings in synovial fluid, classified according to specific joint diseases

Disease

Type

Color
Amount (mL)
pH

Viscosity
Mucin

Cell per μL

Osteo­arthritis

Irritant
effusion

Amber, clear

> 3.5

7.54–7.25

High

Good

Leukocytes
< 2,000

Lymphocytes
75%

Rheumatoid
arthritis

Inflam.

Yellow,
greenish

Clear, turbid

Up to 80

7.41–6.85

Strongly
decreased

Moderate,
poor

Leukocytes
up to 60,000

Psoriatic
arthritis

Inflam.

Lemon yellow,
Green

Turbid

> 3.5

Low

Moderate

Leukocytes
5,000–40,000

PMN > 60%

Ankylosing
spondylitis

Inflam.

Yellow, Clear

> 3.5

Low

Moderate

Leukocytes
up to 10,000

PMN up to 70%

Reiter’s
syndrome

Inflam.

Yellow, purulent

Turbid

> 3.5

Low

Moderate

Moderate

Leukocytes
2,000–50,000

PMN, later on
lymphocytes

Connective
ttissue disease,
SLE

Inflam.

Yellow

Clear, turbid

> 3.5

Normal

Good, moderate

Leukocytes
2,000–10,000

Lymphocytes
up to 50%

Uric acid
induced arthritis
(gout)

Inflam.

Yellow

Milky, turbid,
opaque

> 3.5

Low

Moderate

Leukocytes
> 5,000

PMN > 75%

Chondro-
calcinosis
(pseudo-gout)

Inflam.

Yellow

Milky, turbid

> 3.5

Low

Moderate

Leukocytes
> 6,000

PMN > 50%

Infective
arthritis

Inflam.

Grayish, grayish
yellow

Turbid

> 3.5

Low,
strongly
decreased

Poor

Leukocytes
> 60,000
– 400,000

PMN > 90%

Traumatic joint

Irritant
effusion

Yellow,
sanguineous

Clear, turbid

High

Good

Hemorrhagic
effusion

Irritant
effusion

Yellow,
sanguineous

Clear, turbid

High

Good

Hemarthrosis
(ruptured)

Sanguineous

Turbid, opaque

High

Good

Erythrocytes

Lipo-
hemarthrosis

Sanguineous

Turbid, opaque

High

Good

Table 49-9 Clinical and laboratory findings in inflammatory arthritis

Septic arthritis

The septic arthritis includes all joint infections caused by pyogenic bacteria. The inflammatory process in acute septic arthritis starts within the synovium or, the fluid of a joint becomes the culture medium for bacteria. The route of entry can be /46/:

  • Hematogenous spread by the lodgement of pathogens in the synovial capillaries
  • Spread from contiguous infected foci, such as epiphyseal or subchondral osteomyelitis
  • Spread of a focus of infection elsewhere, such as otitis media or a furuncle or impetigo may reveal septic arthritis in children. In any infant with septicemia, involvement of the hip joint must be suspected
  • A painful joint after arthroscopy, or arthrography in adults with a subacute presentation.

Non-gonococcal septic arthritis usually involves one joint, most commonly the knee. Most patients who suffer such infections have one or more underlying disorders that may be a risk factor /46/.

According to a study in New Zealand the overall incidence of septic arthritis in approximately 6 cases per 100.000 population per year. The incidence is age dependent, with the highest incidence under 15 and over 55 years. The joint most commonly involved in infants is the hip, in older children it is the knee.

Synovial fluid: Cell count > 50 × 109/l and > 90% PMN are indicators of pyogen infection. The concentration of glucose is decreased, the levei of lactate is increased.

Viral arthritis

Viral infection sometimes proceeds to the manifestation of an organ specific inflammation and may be the pathogenic factor of arthritis, particularly of rheumatoid arthritis. Virus associated arthritis seems to be caused to one or the combination of the following mechanisms  /47/:

  • Specific or non-specific modulation of the host’s immune system by virus
  • Viral infection induces novel or aberrant expression of autoantigen
  • Viral antigen mimics auto antigen: cross reactivity results in autoimmunity
  • Viral induced synovial inflammation: direct modulation of gene expression in the target cells by the virus.

Arthritis syndromes with specific viral infections can be associated with Human T cell lymphotropic virus type 1, Parvovirus B19, Hepatitis C virus and Rubella virus. When arthritis and a positive antibody titre (especially of the IgM class in the acute stage) against a distinct virus are found together in a patient, the possibility of a diagnosis of virus associated arthritis should be considered /47/.

Synovial fluid: Detection of viral particles or gene products in affected joints is a key to making final diagnosis /47/.

Reactive Arthritis

Reactive arthritis is classically seen following infection with enteric pathogens such as Yersinia, Campylobacter and Shigella or following urogenital infection with Chlamydia, Mycoplasma and Ureaplasma (Reiter’s syndrome). The acute episode is a few days to weeks ago /48/.

Reactive arthritis is usually an asymmetrical oligoarthritis, generally involving less than 6 joints, with a tendency to affect the lower limbs. However, every joint can be affected and a proportion of patients have a monoarthritis. The affected joints become rapidly hot and swollen, and large effusions can develop in the knee. Septic arthritis or crystal induced arthritis are important differential diagnosis.

The direct detection of enteral pathogens is usually no longer possible. An attempt to detect Chlamydia, Mycoplasma and Ureaplasma is worthwile /49/. Signs of inflammation in acute disease are the elevation of CRP (100–200 mg/L) and leukocytosis. Extra articular manifestations are most frequently associated with more severe disease, a less favorable prognosis and HLA-B27 positivity.

Synovial fluid: It only makes sense to differentiate it from septic arthritis because a pathogen is culturally not detectable in reactive arthritis.

Lyme disease, articular manifestations

In the USA, arthritis is the predominant manifestation of disseminated B. burgdorferi infection. About 60% of patients develop joint manifestations weeks to years after the initial infection. In Europe a smaller proportion of patients experience arthritis. The course of arthritis is usually intermittent with acute attacks and transient seeming remissions. Involvement of the sacroiliac joints or the spine does not occur in Lyme borreliosis. Lyme arthritis cannot be distinguished from reactive arthritis or other acutely presenting oligoarticular diseases on clinical grounds alone /50/.

In some cases there is no overt arthritis, and the disease is characterized by non specific arthralgias, myalgias and periarticular pain.

Synovial fluid: Cell count 25 × 109/L, predominantly PMN. Antibody screening in serum is recommended. PCR analysis of the joint fluid and synovial biopsies may be helpful.

Gonococcal arthritis

Gonococcal arthritis results from blood dissemination of N. gonorrhoeae from primary sexually acquired mucosal infection. The infection is believed to develop in 0,5–3% of patients with mucosal infection. Gonococcal arthritis is mainly, but not exclusively, observed in sexually active young adults. The male/female sex ratio is 1 : 3 or 1 : 4 in most series.

Asymmetric, severe poly arthralgia, which may be migratory or additive, peaks within a few days and may resolve spontaneously. Fever, usually of moderate intensity, and chills are common. The arthritis is observed in less than 50% of cases. Mono articular involvement is more frequent than polyarthritis. Any joint may be affected, the most commonly involved being the knees, wrists, ankles and finger joints. Hip involvement is rare /51/.

Synovial fluid: Leukocyte count (10–100) × 109/L, intracellular and extracellular location of gonococci are observed in Gram stain. Positive culture in synovial fluid approximately 50% of cases, more often positive from the mucosal of entry. PCR identification of N. gonorrhoeae in the synovial fluid; diagnostic sensitivity 96–98%, specificity 78–80% /52/.

Osteoarthritis

Osteoarthritis (OA) is characterized as a multi-disease with inflammation, immune and central nervous system dysfunction playing central roles in whole joint damage, injury progression, pain and disability. It is distinguished from rheumatoid arthritis by the hypertrophic changes in the subchondral bone seen on radiographs /53/. The pathognomonic signs of osteoarthritis on radiographs are joint space narrowing, osteophytes, subchondral sclerosis and subchondral cysts. The usual initiator of subchondral bone remodeling is abnormal mechanical loads. These lead to micro fractures and what may be a fracture healing response. Coupled with articular cartilage fissuring and loss, the synovial fluid and its associated cytokines can directly infiltrate the subchondral bone and affect all the cells present.

Inflammation in patients with failed joint replacements

Whilst the majority of patients have a successful outcome, with reduced pain and improved function, a proportion of 10–20% develop problems including joint stiffness, reduced range of motion and pain. Chronic pain and restricted motion are a devastating complication following total knee replacement. Classification of septic prosthetic failure is based on the pathogenicity and etiology of the infection. Accordingly; infection is classified as follows /54/

  • Acute peri operative infection with early post operative onset and highly virulent bacteria
  • Primary chronic low grade infection with delayed onset and low virulence or small colony forming bacterial stains
  • Late hematogenous high grade infection.

While the diagnosis of acute infection and late hematogenous infection is easy given the typical clinical and laboratory presentation, chronic low grade infection is challenging to detect. Quantitative measurement of alpha defensin plus CRP seems to be the most helpful combination to distinguish between aseptic loosening and low grade joint infection. Synovial fluid CRP > 2 mg/L and alpha defensin > 90.000 pg/mL identified 9 of 11 patients with low grade infection /55/.

Synovial fluid crystals – Generally

Chrystal detection and identification play an important role in synovial fluid analysis. The presence of crystals leads to acute inflammation, producing increased leukocytes and a neutrophil- predominant infiltrate. Crystals develop in synovial fluid for one of the following reasons /56/:

  • Cristallization of an elevated plasma constituent that becomes highly concentrated in the joint.
  • Formation of crystals from a degenerative process involving cartilage or bone calcification
  • Introduction of a substance, such as corticosteroids, directly into the joint space.
  • Gout and pseudogout comprise the two major crystal-induced arthritides.

Calcium pyrophosphate dihydrate (CPPD) and hydroxyapatite crytals are the most common calcium containing crystals associated with joint and periarticular disorders /57/. Joint aspiration is helpful to elucidate the afore mentioned differential diagnosis and it should be performed promptly.

Birefringence procedure for crystal analysis using a polarizing microscope: the microscope is equipped with two identical dicrotic or Polaroid filters. One filter, placed in a fixed position above the specimen and below the eyepieces, is called the analyzer. The second filter, placed in a rotating collar below the specimen and above the light source, is the polarizer. Each filter allows light of only one direction to pass. If the bottom filter is rotated until it is aligned 90 degrees in relation to the top filter (crossed), the light coming through the polarizer is blocked by the analyzer so a dark field is seen by the viewer. A specimen containing a birefringent crystal is introduced. Placed in the light path above the polarizer, the crystal refracts the light, allowing it to pass through the analyzer, and appears as a shiny object against the black background. Monosodium urate crystals usually are seen in the form of medium-size rods or needles, whereas CPPD crystal often are small and shaped like a diamond, rhomboid, or square. The description is a copy of Ref. /56/.

– Gout

The solubility of uric acid in blood and tissue is decisive for the development of gout. The monosodium urate crystals only form when the solubility product is exceeded. At physiological pH and normal body temperature this can occur at uric acid levels > 7 mg/dl (0.4 mmol/l). At acid pH or lower temperature the solubility product of monosodium urate is lower /58/. After obtaining synovial fluid the intra- and extracellular needle shaped uric acid crystals are identified under microscope in compensated polarized light from their shape and refraction and distinguished from other crystal arthropathies /59/.

The 2018 updated Europen League Against Rheumatism evidence-based recommendations for the diagnosis of gout /60/ search for crystals in synovial fluid or tophus aspirates is recommended in every person with suspected gout, because demonstration of monosodium urate crystals allows a definitive diagnosis of gout.

– Pseudogout

Specific identification of calcium pyrophosphate dihydrate (CPPD) crystals (Ca2 P2 O7 H2O) in synovial fluid allows the clinician to differentiate between CPPD crystal deposition disease and other inflammatory and degenerative arthritides /61/. The term pseudogout refers to acute, gout like attacks of inflammation that occur in some persons with CPPD deposition disease.

Acute pseudogout is an inflammatory disorder manifest by joint effusions and symptoms and signs of articular inflammation in one or more joints. Patients experience pain, stiffness, and swelling in the affected joint. The self-limited attacks can be abrupt in onset and as severe as acute gout. Pseudogout is more common in large than in small joints. The knee is the most commonly involved joint. Diagnosis of pseudogout is made by identifying CPPD crystals by polarizing light microscopy in the synovial fluid of affected joints. The weak birefringerence of CPPD crystals render them more difficult to discern than monosodium urate crystals. CPPD crystals can be quite sparse in number. Conditions strongly associated with CPPD crystal deposition are age, previous joint surgery, osteoarthritis, trauma, gout, hyperparathyroidism, hemochromatosis, hypophosphatasia, and hypomagnesemia.

– Basic calcium phosphate crystal disease

Basic calcium phosphate (BCP) crystals are predominantly composed of partly carbonate substituted hydroxyapatite, but also include octacalcium phosphate and magnesium whitlockite /62/. Crystals are identified using alizarin red S histochemical staining of synovial fluid from the affected joint. The shoulder is the joint most commonly associated with BCP crystal periarthritis but BCP periarthritis involving the distal interphalangeal joints in a patient with SLE is reported /63/.

Intra articular BCP crystal deposition is associated with osteoarthritis, and Milwaukee shoulder syndrome, but also with connective tissue disease, including dermatomyositis, scleroderma and systemic lupus erythematosus. One of the earliest cellular effects of BCP crystals is a bimodal increase in intracellular calcium. The key mechanisms whereby BCP crystals may cause tissue damage are the induction of mitogens, the upregulation of matrix metalloproteinase production, the stimulation of cyclooxygenases and the prostaglandin production /62/.

Figure 49-1 Algorithm for synovial fluid investigation /612/.

Aspirate Heparinized SF Native SF Simple visualexamination < 6000/μl < 6000/μl > 50.000/μl Cell count MicroscopyRagocytesCrystals Bacteriology(10–15 mL native SF) Cultures Differential blood count Gram stain 10 min centrifugation at 1,000–3,000 rpm Sediment Supernatant (hyaluronidase) Differentialblood count Gram stain Clinical chemistryexaminations Immunologicalexaminations

Figure 49-2 Intervals of leukocyte counts associated with the different SF types. Cell count per μL

10 2 normal non-inflammatory inflammatory 10 3 10 4 10 5 septic
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