Ordering and interpreting rheumatologic laboratory tests for patients with inflammatory symptoms can be challenging. Having conceptual guidelines and indications to effectively order serologic tests can help to avoid unnecessary testing and potentially confusing results. The tests should help confirm a clinical impression or sort out a differential diagnosis. Indiscriminate ordering or asking for the “arthritis panel” should be avoided. Many rheumatologic illnesses are relatively rare, and because all tests have falsepositive rates, a positive result in a patient with a low pretest probability of having the illness is likely a false-positive.
Acute Phase Reactants
Erythrocyte Sedimentation Rate
The erythrocyte sedimentation rate (ESR) is a widely used, nonspecific, indirect measure of systemic inflammation. The Westergren method, the current accepted method for performing the ESR, uses a 200-mm vertically aligned column with a 2.5-mm diameter. The column is filled with blood anticoagulated with K3 EDTA.1 The distance that the column of red blood cells settles within the plasma in 1 hour is reported in mm/h.
The factors affecting the sedimentation of red blood cells include the size of the cells, the viscosity of the plasma, and the repellent forces between the negatively charged sialic acid molecules on the surface of each red blood cell (Fig. 1). The presence of large or positively charged asymmetric proteins, especially fibrinogen but also immunoglobulins, counteracts the repellent force and allows the formation of rouleaux, or coin-like rolls of cells, causing the cells to settle more rapidly than usual.2 Many factors influence the ESR (Table 1). The range of normal in many laboratories is 0 to 15 mm/h for men and 0 to 20 mm/h for women. The ESR increases with age, and to avoid unnecessary concern, it has been suggested that age divided by 2 for men and age plus 10 divided by 2 for women be used as the upper limits of normal.3 Besides age, the other important factor that elevates the ESR is obesity.4 Adipose tissue is a source of interleukin-6 (IL-6), which increases liver production of acute phase reactants, including fibrinogen and C-reactive protein (CRP).5
Although widely used, the ESR has limited application as a diagnostic test, and it must always be interpreted in a clinical context. Sox and Liang2 have pointed out that, for patients with vague symptoms who appear to be normal on examination, fewer than 6 in 10,000 benefit from an ESR test. In addition, a sample of patients from Scandinavia with unexplained elevation in the ESR followed over time rarely developed any serious illness.6 The following are general guidelines:
(1) The ESR may take a few days to a week to elevate and a similar amount of time to regress once the inflammatory stimulus is gone.
(2) An ESR is helpful to confirm a clinical impression regarding the presence or absence of inflammatory disease, although occasionally patients with inflammatory diseases present with a normal ESR. Age and obesity elevate the ESR.
(3) Resolution of the ESR is a useful marker of treatment success in illnesses such as rheumatoid arthritis, septic arthritis, and osteomyelitis.
(4) A very high ESR (>100 mm/h) almost always is associated with underlying pathology.
First discovered in 1930 by Tillet and Frances, CRP owes its name to the ability of this liver-derived protein to precipitate pneumococcal Cpolysaccharide in the presence of calcium.7 The liver produces CRP under the influence of the inflammatory cytokines IL-1 and IL-6. CRP levels begin to rise within 6 hours of an inflammatory stimulus, peak in approximately 50 hours,8 and fall rapidly once the stimulus is removed. CRP thus provides a more immediate picture of the level of inflammation than does the ESR, in which the fibrinogen level rises and falls more slowly9 (Fig. 2). Its function in vivo is thought to be to assist in the activation of the complement system, influence phagocytic cell function, and augment cell-mediated cytotoxicity to amplify the immune response.8
The normal range of the CRP level is 0 to 1 mg/dL. A level between 1 and 10 mg/dL is considered a moderate elevation and a level above 10 mg/dL, a marked elevation. Elevations are seen not only in expected situations, such as with infectious, inflammatory, and malignant diseases, but also with pregnancy and trauma. Factors such as age, sex, anemia, and red blood cell shape have little effect on the CRP level, so there may be a lower false-positive rate than occurs with the ESR. The CRP level is a direct measure of inflammation and is as useful as the ESR in most situations. Since CRP is becoming easier and cheaper to assess, it may someday supersede the ESR because it more accurately reflects the current level of inflammation.
Rheumatoid factor (RF) as measured clinically is an IgM antibody directed against the Fc portion of the patient's own IgG (Fig. 3). Why the body makes RF is uncertain, although it may be to help clear immune complexes by stabilizing low-affinity IgG-antigen interactions and improving opsonization by fixing complement more efficiently than would occur without RF.10 RF is commonly secreted during acute infections and probably is part of the normal immune response. Other isotypes of RF can be produced (IgG, IgA) but currently are not measured clinically. However, IgA RF may prove to be useful clinically because it has been shown to help predict a more severe disease course.11
Latex agglutination testing, usually done by hand, is widely used to measure RF, although it is being supplanted by other methods. Enzyme-linked immunosorbent assay (ELISA) and nephelometry are capable of being automated and are more sensitive than the latex method.11 The latex test is reported in a titer, that is, serial dilution of serum, which is a discontinuous measurement and has an accuracy of ±1 dilution. Most clinical laboratories consider a dilution >1:40 to be positive. Nephelometry is able to quantitate antigen-antibody interaction by measuring laser light scatter caused by the formation of immune complexes. The nephelometry test is usually reported in international units (IU); the normal range depends on the specific laboratory but usually is <20 IU.
When RF testing is ordered indiscriminately, the false-positive rate can be quite high. In one study, only 86 of 563 RF tests ordered during a 6-month period were positive, and only 21 of the 86 patients actually had rheumatoid arthritis.12 Thus, three quarters of positive results were false-positive for the diagnosis of rheumatoid arthritis. A number of critical points should be kept in mind when ordering RF tests:
(1) RF is present in 70% to 90% of patients with rheumatoid arthritis; therefore, a negative RF result does not rule out rheumatoid arthritis.11
(2) RF may take several months to appear in the serum after arthritis develops.
(3) The level of RF is prognostic, that is, the higher the level, the worse the prognosis.
(4) An elevated RF level is not specific for rheumatoid arthritis and can be found in a variety of other illnesses, including other rheumatologic diseases (eg, lupus erythematosus, Sjögren's syndrome, myositis, and cryoglobulinemia) as well as infectious diseases (eg, endocarditis, tuberculosis, syphilis, and hepatitis C). An elevated RF level also is associated with aging, idiopathic pulmonary fibrosis, cirrhosis, and sarcoidosis. This fact is particularly important because diseases like hepatitis C can present with rheumatoid-like synovitis and a positive RF test result.
Diagnostic testing for systemic lupus erythematosus (SLE) began in 1948 with the description of the lupus erythematosus cell, a polymorphonuclear neutrophil that had engulfed a cell nucleus. The lupus erythematosus cell test is now of historical significance and is no longer available at most immunology laboratories. In 1957, Friou described the fluorescent antinuclear antibody (FANA) test, which marked a new era in the diagnostic testing of SLE.13 Over time, subsets of antinuclear antibodies (ANAs) have been used to classify various autoimmune syndromes.
ANA testing is done in two sequential steps. The first is testing for the presence of a FANA; the second, if the FANA test is positive, is testing for specific autoantibodies that help to classify the potential underlying connective tissue disease.13,14 The FANA test is done by placing patient sera over a thin layer of cells from a HEp-2 cell line (a line of human epithelioma cells) (Fig. 4). ANAs attach to the specific nuclear antigen in the cell nucleus and will not wash away. An antihuman IgG conjugated to a fluorochrome tag is then added to the cells, and the cells are viewed under a microscope with ultraviolet light excitation. Common patterns include speckled, rim, homogeneous, centromere, and nucleolar, reflecting the types of antigens present, which have a loose association with specific syndromes (Table 2).13 If the FANA test is positive, a more specific test, such as an ELISA, can be done to detect specific antinuclear antigens. The ELISA results help to classify the autoimmune disorder. Most laboratories report a FANA titer of 1:40 to 1:80 as positive, although patients with an active autoimmune disorder rarely have such low titers. Most rheumatologists view titers ≥1:160 as significant. The most helpful result on the FANA test is a negative one. A positive test does not mean the patient has an autoimmune condition but indicates that clinical correlation is needed. The following points may be useful when interpreting ANA test results:
(1) Ninety-five percent of patients with SLE have a positive FANA test result; the remaining 5% have a negative FANA test result but a positive result for antibodies to SS-A (Sjögren's syndrome A, also known as antibodies to Ro). Thus, negative FANA and negative SS-A test results rule out SLE.
(2) A positive FANA test result does not mean a disease is present. Low titers (eg, 1:40, 1:80) in patients without objective abnormalities (aches and pains, fatigue) are likely false positive.15
(3) A diagnosis of SLE is made on the basis of a positive FANA test result at a reasonable titer and the presence of three other objective criteria for SLE, such as swollen joints, nephritis, or pericarditis (not aches and pains or reports of a rash in the distant past).
(4) Other diseases can cause a positive FANA test result, notably diseases of the thyroid, such as Graves' disease or Hashimoto's thyroiditis. The false-positive rate increases with age, as do false-positive rates for many other autoantibodies.
Several specific patterns of autoantibodies are useful to remember:
(1) A centromere pattern on the FANA test indicates CREST syndrome (calcinosis, Raynaud's phenomenon, esophageal problems, sclerodactyly, telangiectasias).
(2) Antibodies to dsDNA (doublestranded DNA) or Sm (Smith) are seen in SLE.
(3) Antibodies to Scl-70 are seen in a minority of persons with scleroderma but predict severe disease.
(4) Antibodies to histone are seen primarily in persons with druginduced SLE.
(5) High levels of antibodies to ribonucleoprotein are seen in mixed connective tissue disease, a condition that has features of scleroderma and SLE.
Anticardiolipin Antibodies and Lupus Anticoagulant
These tests are used to evaluate patients with hypercoagulable states. Anticardiolipin (or antiphospholipid) antibodies are a heterogeneous family of antibodies that are directed against components of the coagulation pathway. Their presence may put patients at risk of thrombosis. The same is true of the lupus anticoagulant antibodies, which prolong the partial thromboplastin time and also may lead to thrombosis.
These two kinds of antibodies can be present in up to 40% of patients with SLE or can occur in isolation, leading to primary antiphospholipid syndrome. Clinical manifestations of these antibodies include recurrent fetal loss in the second trimester, venous and arterial thrombosis, and thrombocytopenia. 16 Other possible manifestations include transverse myelopathy, cardiac valvular disease, and osteonecrosis. Three points should be kept in mind when considering whether testing will be useful:
(1) Anticardiolipin antibodies and lupus anticoagulant are not diagnostic tests for SLE or other connective tissue diseases.
(2) If hypercoagulability is a concern, testing should be done for both lupus anticoagulant and anticardiolipin antibodies because a patient may have one or both. These tests are only part of an evaluation for a hypercoagulable state.
(3) These tests should be ordered only when hypercoagulability is strongly suspected. Like all other such tests, they have a relatively constant false-positive rate and could lead to unwarranted concern and expensive follow-up tests.
Human Leukocyte Antigen-B27
The human leukocyte antigen (HLA) molecules are part of the major histocompatibility complex encoded by genes on the short arm of chromosome 6. These molecules are important in self-recognition and immune response. HLA-B27 is one of the class I antigens that are found on all cells except red blood cells and are important for cell-mediated immunity. Certain HLA molecules have been found to indicate risk of various diseases. The HLA-B27 antigen indicates a risk of spondyloarthropathies.17 This antigen is found in approximately 8% of European-Americans and 4% of African-Americans. The test has a 92% sensitivity and specificity for ankylosing spondylitis in EuropeanAmericans and is present in >90% of affected individuals. The test also is useful for diagnosing Reiter's syndrome because 80% of these patients carry the antigen.
The diagnosis of ankylosing spondylitis depends on the history, examination, and typical radiographic findings of sacroiliitis. When the history and examination are highly suggestive but there is no radiographic evidence of the disease (either on plain radiographs or computed tomography [CT]), HLA-B27 may be helpful in decision-making. In these borderline situations, a positive test makes the likelihood of the disease approximately equal to the specificity (ie, about a 92% chance of having ankylosing spondylitis), whereas a negative test makes the likelihood of not having the disease approximately equal to the sensitivity (ie, about a 92% chance of not having ankylosing spondylitis).16
Generally, an HLA-B27 test should be ordered only when the patient's history is compatible with ankylosing spondylitis or Reiter's syndrome and definitive radiographic changes are lacking, or, in the case of Reiter's syndrome, when clinical features are insufficient to make a definitive diagnosis. The test should not be ordered when the history clearly is that of mechanical low back pain because <20% of HLA-B27-positive patients have associated clinical manifestations.
Prolonged hyperuricemia frequently leads to the clinical symptoms of gout. In patients with arthritis consistent with gout, demonstrating the presence of intracellular crystals is key to the diagnosis. Serum uric acid levels may be elevated during an acute attack of gout, but in some patients, the levels may decrease 1 to 2 mg/dL or more while inflammation is present. Thus, in a patient with gout with a preattack serum uric acid level of 7 to 9 mg/dL, that elevated level may fall into the normal range during an acute attack.18 This is particularly true in alcoholics.19 Uric acid levels are best checked once the attack has subsided. Demonstrating uric acid crystals from a joint aspiration is not necessary with each attack but should be done at least once to aid decision-making.
The diagnosis of gout sometimes is assigned incorrectly, especially to patients with joint pain and asymptomatic hyperuricemia but without a convincing history of gout.20 Therefore, the diagnosis rests on visualizing urate crystals in white blood cells taken from the inflamed joint, not simply on the presence of hyperuricemia. Only a small amount of fluid (a drop or less) is necessary to make the diagnosis. Because gout can mimic septic arthritis, aggressive therapy for infection should be avoided before gout has been ruled out.
Lyme arthritis is a late manifestation of infection with the tickborne organism Borrelia burgdorferi. The disease most commonly occurs in the spring and summer in the northeast and upper midwest of the United States.21 Arthritis occurs either as an intermittent, migratory polyarthritis in half of the patients who develop late-stage disease or as a chronic monoarthritis, usually affecting the knee with large, inflammatory effusions.
The diagnosis should be made on clinical grounds (the season, regional location, and pattern of the rash— erythema chronicum migrans). Positive serologic testing increases the posttest probability of Lyme disease and helps in deciding on antibiotic therapy. Testing detects the presence of antibodies to the causative organism, not the organism itself, so those who may have been infected but have cleared the organism still would test positive but may not have active disease. In addition, the test has a 5% false-positive rate, and false-positive results are common among persons with other forms of arthritis, such as rheumatoid arthritis and SLE. The initial test is an ELISA; if it is positive, it is followed by a confirmatory Western blot analysis. In addition, polymerase chain reaction (PCR) testing can detect B burgdorferi DNA within the joint fluid of patients suspected to have Lyme arthritis.21 This test has a high specificity and sensitivity for the organism and may be useful in distinguishing patients with active B burgdorferi infections from those who are simply seropositive and have other causes of arthritis. Patients with late Lyme disease (ie, those with arthritis) almost always are seropositive, so a negative test virtually rules out Lyme arthritis.
Lyme serology should be ordered only when the clinical diagnosis is highly suggestive. Using this test for evaluating patients with a low probability of infection can lead to incorrect diagnosis and overtreatment. In high-risk areas, a certain percentage of the population will be antibody-positive but disease-negative, so clinical correlation is particularly important. In this situation, PCR for B burgdorferi in the joint fluid may be useful.
Carpal Tunnel Syndrome and Stiff Joints
A 26-year-old woman was referred for evaluation and treatment of carpal tunnel syndrome. She reported paresthesias in both hands, especially the right, that awakened her from sleep and also were present while driving. She also reported stiffness of 2 months' duration in the hands and wrists that was worse in the morning than during the day.
The physical examination disclosed swelling and tenderness of the metacarpophalangeal, proximal interphalangeal, metatarsophalangeal, and wrist joints. She had positive Tinel's and Phalen's signs, greater on the right than left. Otherwise, the examination was normal. Laboratory testing included a normal complete blood count, an ESR of 42 mm/h, a FANA titer of 1:40, and negative results for SS-A and hepatitis C antibodies. RF was elevated at 215 IU.
She was given neutral wrist splints and referred with the diagnosis of RA to a rheumatologist. She was given methotrexate and lowdose prednisone. In spite of this therapy, she required endoscopic carpal tunnel release on the right to relieve symptoms. Her rheumatoid arthritis currently is well controlled by methotrexate 15 mg/week and prednisone 2.5 mg/day.
This patient presented with an inflammatory polyarthritis affecting small joints of the hands and feet, with carpal tunnel syndrome as a result of underlying synovitis. The initial differential diagnosis included RA, lupus, and hepatitis C infection. A diagnosis of RA was made on the basis of clinical and laboratory data. Typically, a FANA titer of 1:40 is not clinically meaningful.
Low Back Pain With a Sore Heel
A 25-year-old man was referred for persistent right Achilles tendinitis. The tendinitis had appeared 2 months earlier and possibly began after a game of soccer. He had tried nonsteroidal anti-inflammatory drugs (NSAIDs), rest, and a heel lift, all without benefit.
On physical examination, he had trouble removing his socks because of back stiffness. There was tenderness to palpation of the sacroiliac joints bilaterally and also low back pain on both sides with Patrick's test. Lumbar range of motion was normal. There was also tenderness and swelling of the right Achilles tendon at the insertion into the calcaneus. A plain radiograph, pelvic outlet view, demonstrated sacroiliitis consistent with ankylosing spondylitis. His ESR was elevated at 45 mm/h. He was referred to a rheumatologist and given high-dose NSAIDs and sulfasalazine; the symptoms resolved.
Insertional Achilles tendinitis is often caused by a spondyloarthropathy. This, together with back stiffness, suggests ankylosing spondylitis. If the radiograph of the sacroiliac joints had been negative or equivocal, CT of the sacroiliac joints could have been done. If this were negative, an HLA-B27 test could have been considered. Testing for RA and SLE was not needed because Achilles tendinitis is unusual in these diseases.
Arthralgias and Fatigue
A 36-year-old woman reported bilateral hand pain and numbness. She was referred for possible carpal tunnel syndrome. She had developed the pain in the hands several years earlier and had recently quit her job as a secretary because of her symptoms. She also reported fatigue and diffuse muscle aches.
The physical examination was remarkable for tenderness without swelling of multiple metacarpophalangeal, proximal interphalangeal, and distal interphalangeal joints of both hands, with grimacing. She had tenderness around various joints and muscles to the pressure of the examination, without evidence of swelling. Neurologic examination was unremarkable except for Tinel's sign at the right wrist, which produced pain into all of the fingers. Review of previous laboratory tests for her complaints demonstrated a normal complete blood count, chemistry panel, ESR, and thyroid-stimulating hormone, calcium, and creatine kinase levels. Electrodiagnostic study results were normal. She was referred to a rheumatologist. A diagnosis of fibromyalgia was made, and she was prescribed lowdose nortriptyline and an exercise program.
Her presentation is not unusual for fibromyalgia. RF, FANA, and SSA tests should be avoided in such situations because they confuse the issue. Many patients with fibromyalgia have been told that they have SLE, based on a false-positive FANA test result.
Bilateral Shoulder Pain
A 66-year-old woman was referred to an orthopaedic clinic for persistent rotator cuff tendinitis. The symptoms had begun 4 months earlier. She had stiffness and aching in both shoulders with overhead activities. Morning stiffness in the shoulders and hips required her to roll out of bed. She had completed two courses of physical therapy for the shoulder, without much benefit. After her latest physical therapy course, her primary care physician had given her a corticosteroid injection in the right shoulder, which improved her general stiffness and pain for 4 days, although both then returned.
The physical examination revealed signs of rotator cuff tendinitis bilaterally. The rest of the examination was normal; in particular, there was no evidence of synovitis of the small joints of the hands or feet. Laboratory tests demonstrated a hematocrit level of 33%, normal chemistry panel, and ESR of 80 mm/h. A suspected diagnosis of polymyalgia rheumatica was confirmed by a rheumatology consultant. Low-dose prednisone was started, and her symptoms resolved dramatically within 24 hours.
The distribution of symptoms and signs suggests the diagnosis of polymyalgia rheumatica. RF and FANA tests are not needed unless there is joint swelling or other objective abnormalities. The arthritis/ periarthritis typical of polymyalgia often can cause rotator cuff signs and symptoms. In any older person with bilateral shoulder symptoms, polymyalgia rheumatica should be considered in the differential diagnosis.
Swollen Knee and Inflammatory Fluid
A 55-year-old man reported swelling of 1 month's duration in the left knee. His history was negative for trauma or previous problems with the knee. He had stiffness in the morning, with some improvement during activity.
The physical examination revealed a moderate-sized effusion of the knee. There was diffuse swelling of the left second toe and a patch of psoriasis on his scalp. Aspiration of the joint yielded 40 mL of cloudy fluid with a white blood cell count of 30,000/μL, mostly neutrophils. Laboratory tests demonstrated a normal complete blood count and an ESR of 35 mm/h.
The diagnosis of psoriatic arthritis was considered; he was prescribed NSAIDs and referred to a rheumatologist, who prescribed methotrexate. There was moderate improvement of the joints and skin. Etanercept was added, with notable improvement.
This patient had an inflammatory form of arthritis confirmed by the fluid in the knee. Checking the synovial fluid cell count at the knee may help avoid arthroscopy. The presentation in this case is typical of psoriatic arthritis, and there is no need for FANA or RF tests. A follow-up ESR or CRP test is reasonable as a marker of treatment success. Psoriatic arthritis is not associated with HLAB27, and testing would not be useful in this situation. In an endemic area, Lyme serology is not unreasonable when only the knee is affected.
Bilateral Heel Pain
A 24-year-old soccer player reported bilateral heel pain of 6 months' duration that was worse in the morning and partially improved with activity. He had tried NSAIDs (with some benefit), over-thecounter orthotics, and even a posterior night splint, without a notable change in symptoms. He also reported occasional low back stiffness that he ascribed to his soccer play. He was diagnosed with bilateral plantar fasciitis and referred for other therapeutic options.
Examination demonstrated evidence of bilateral plantar fasciitis and was otherwise negative. Plain radiographs, including a pelvic outlet view of the sacroiliac joints and views of the feet, were unremarkable. The CRP level was elevated at 2.8 mg/dL, and results of subsequent CT of the sacroiliac joints were negative. HLA-B27 antigen was then checked and was positive.
He was referred to a rheumatologist and begun on indomethacin and sulfasalazine, with excellent response. His subsequent course was marked by intermittent bouts of iritis, and 2 years later, radiography revealed that he had developed sacroiliitis.
An HLA-B27 determination is useful in this situation, that is, negative radiographic results but positive symptoms and an elevated CRP level. HLA-B27 antigen testing was helpful because of the impression that the patient had an inflammatory form of enthesopathy. The HLA-B27 test also aided in treatment decisions. The CRP level was useful to confirm the inflammatory nature of the symptoms, but it would not have ruled out an inflammatory cause if the level had been normal.
A 45-year-old forest ranger who worked in Minnesota was referred for persistent right knee swelling. He was known to be antibody positive for B burgdorferi. One year earlier, his right knee had swelled, with the synovial fluid showing 25,000 white blood cells/μL, mostly neutrophils. He had been treated with 60 days of doxycycline without change in the swelling. Before treatment with doxycycline, PCR results of the synovial fluid were positive for B burgdorferi. He was sent for arthroscopic biopsy to rule out granulomatous disease and to obtain synovium for B burgdorferi culture.
The physical examination revealed only the swollen right knee. A repeat synovial PCR for B burgdorferi was negative, and the patient was thought to have post-Lyme inflammatory arthritis. A right knee synovectomy was done, and he was given hydroxychloroquine, which controlled his synovitis.
This is a case in which persistent synovitis developed after Lyme arthritis. Many such patients carry the HLA-DR4 gene and require treatment with disease-modifying antirheumatic drugs, such as hydroxychloroquine, as well as reduction of the synovial mass by arthroscopic synovectomy. Without such treatment, some patients develop erosive arthritis.
Understanding the basic principles of tests for inflammatory joint disorders can help the clinician distinguish various forms of synovitis. Testing should be ordered only after a thorough history and physical examination. The selection of tests should be specific to help clarify a borderline clinical scenario. Nonselective use of testing may lead to false-positive results, which can cause unnecessary concern and increased expense.
We wish to thank Mart Mannik, MD, for his thoughtful review and useful suggestions.