Laboratory Tests in Rheumatology

Laboratory Tests in Rheumatology

Authors:
Saru Sachdeva, MD, Fellow of Rheumatology, Loma Linda University, Loma Linda, CA
Keith Colburn, MD, FACP, FACR Professor of Medicine, Chief of Rheumatology, Loma Linda University and Loma Linda VA Medical Center, Loma Linda, CA

Peer Reviewer:
Clara L. Carls, DO, Program Director, Family Medicine Residency, Hinsdale, IL

A comprehensive history and thorough physical examination is the best screening test in diagnosis of autoimmune diseases. Blood tests are useful adjuncts in confirming suspected diagnosis. In certain cases they may be useful to monitor disease activity, estimate disease severity, determine response to treatment, and assess prognosis. The pretest probability of a test is an important determinant in the value of a test to diagnose a certain condition. A positive result for a test with high pretest probability helps to confirm diagnosis, whereas a negative test with low pretest probability will help to rule out diagnosis.

The improper use of tests is fraught with pitfalls. A positive test result in an inappropriate clinical setting can cause unnecessary patient anxiety. It can lead to further advanced testing and potentially dangerous treatments. Serial testing, such as ANA in lupus, does not add any new clinical information and increases the overall cost of health care. Another issue is the lack of standardized methods in interpreting results. The reporting range often can be confusing for primary care physicians when laboratories have a low threshold for a positive result. This can lead to a higher number of false positives.

The objective of the article is to discuss laboratory tests commonly used in evaluating rheumatologic diseases and help primary care physicians to evaluate and interpret common positive and negative test results, and therefore lead to more judicious use of available resources. The common laboratory tests discussed in the article are shown in Table 1.

Acute Phase Reactants

Acute phase reactants are plasma proteins whose concentration increases or decreases during inflammatory conditions. The change in concentration should be at least 25% for the test to be of value.¹ Positive reactants increase and negative reactants decrease during inflammatory states. (See Table 2.)

Erythrocyte Sedimentation Rate

Erythrocyte sedimentation rate (ESR) is the most widely used marker for inflammation. ESR is an indirect measure of serum acute phase plasma protein concentrations, especially fibrinogen. The ESR is the measure of the quantity of red blood cells (RBC) that precipitate in a tube in a defined time. This is based on serum protein concentrations and RBC interactions with these proteins. RBC shape and number also have an influence on the rate of the fall. The sample needs to be processed within a few hours to ensure test accuracy.

The ESR rises in inflammatory states. However, high ESR result can be falsely seen in the presence of anemia,² renal disease, and hypergammaglobulinemia. It is also influenced by patient’s age and gender.³ (See Table 3). The ESR can be spuriously low in hemoglobinopathies, polycythemia, and cryoglobulinemia.

The changes in ESR correlate with rheumatoid arthritis (RA) activity, polymyalgia rheumatica (PMR), and giant cell arteritis.⁴ The ESR at the time of PMR diagnosis may have prognostic value. The ESR also may rise with trauma, infection, ischemia, and malignancy. The rate of change in ESR is slow and usually lags behind the decrease in inflammation.

C-Reactive Protein

C-reactive protein (CRP) is an innate immune protein. The role of the protein is to help opsonize pathogens for phagocytosis. It also activates the complement system. The production is under the control of IL-1, IL-6, and TNF-alpha. CRP rises rapidly after inflammation and tissue injury. CRP levels are not subjected to fluctuations secondary to anemia etc., unlike ESR. CRP levels do change with age, gender, and race of the patient. (See Table 3.) CRP levels of < 0.2 mg/dL are considered normal. Levels > 1 mg/dL are indicative of ongoing inflammation.

CRP may be reported as mg/dL or mg/L. Usually, high sensitivity CRP (hsCRP), which detects very low grade inflammation, is reported in mg/L.⁵ hsCRP is a more sensitive CRP test and elevations indicate very low grade inflammation. CRP values between 0.2-1 mg/dL may be present in certain non-inflammatory states (See Table 4). Values > 1 mg/dL are indicative of significant inflammation.

Patients with systemic lupus erythematosus (SLE) tend to have normal or mildly high CRP levels. Elevation of CRP levels in lupus patients may signify infection, vasculitis, serositis, or synovitis. Elevation of CRP in RA has prognostic value. The combination of using both ESR and CRP improves sensitivity of either test alone.

Ferritin

Serum ferritin concentration is influenced by intracellular iron, TNF-alpha, IL-1, IL-6, oxidative stress, and growth factors. This may be elevated in various rheumatologic diseases. Marked elevations in serum ferritin may be seen in Still’s disease and hemophagocytic syndromes. Serum ferritin may be as high 10,000 ng/mL (normal 40-200 ng/mL) in patients with Still’s disease. The levels correlate with disease activity.

Antinuclear Antibodies

Autoantibodies to nuclear antigens are a diverse group of antibodies that react against nuclear, nucleolar, or perinuclear antigens. Antinuclear antibodies (ANA) is commonly associated with autoimmune diseases; however, it is also one of the most inappropriately ordered tests.

ANA is detected using immunoflourescence testing of patient’s serum using cell substrate obtained from human epithelial tumor cell line (Hep 2 cells). Another method is through ELISA. Using Hep 2 cells allows detection of fluorescent pattern (homogenous, diffuse, speckled, peripheral, and rim). (See Table 5.) These patterns may help association with certain autoimmune diseases. However, ELISA is cheaper and more widely available. There is higher incidence of false positives with the ELISA method.

ANA is a highly sensitive test (negative test helps to exclude the diagnosis) and a poorly specific test (positive test does not help in establishing a diagnosis) for SLE and scleroderma (See Table 6). Patients with Raynaud’s phenomenon should have ANA testing done only if signs of connective tissue disease are present. A positive ANA in a patient with Raynaud’s phenomenon increases the risk of developing systemic rheumatic disease from 19%-30%, whereas a negative test reduced this risk to 7%.⁶ Presence of ANA in juvenile idiopathic arthritis patients helps stratify risk for developing uveitis.

Various other diseases (autoimmune, chronic infectious, and malignancy) may be associated with positive ANA (See Table 7).

Studies have shown that 25-30% of healthy controls have positive ANA at lower titers (1:40), 10-15% at 1:80, and < 5% at 1:160. The frequency increases with age, especially in women. In healthy subjects, ANA titer of 1:40 can be seen in 25-30% of relatives of patients with rheumatologic condition.⁷ Therefore, ANA testing should be limited to cases where there is strong clinical suspicion of systemic autoimmune disease. There is also no utility of serial ANA testing in diseases such as SLE and scleroderma.

Types of Antinuclear Antibodies

Anti-dsDNA. The antibodies directed against double-stranded DNA are highly useful in diagnosing SLE. There are four methods to detect dsDNA: Farr assay, Crithidia luciliae assay, ELISA, and flourescent beads. Crithidia assay and ELISA are more commonly used. Crithidia assay uses principle of indirect immunoflourescence by using dsDNA from the hemoflagellate organism. It has a sensitivity of 50-80%. ELISA test is positive in 70-80% patients with SLE. Overall, dsDNA antibody has a sensitivity of 57.3% and specificity of 97.4% in diagnosing SLE.⁸ Less than 1% of patients may have positive dsDNA antibodies in the absence of positive ANA.

Anti-dsDNA antibody titers may correlate with disease activity. In lupus nephritis, anti-dsDNA antibodies tend to increase when disease is active. However, in some patients this may not occur. In such patients, serial testing may not be of much value. Anti-dsDNA antibodies may be found in other conditions (See Table 8).

Anti-Smith and anti-U1RNP antibodies. The Smith and U1 RNP autoantigens localize in small nuclear ribonucleoprotein particles (snRNPs). They are frequently found in patients with SLE. The anti-Smith (anti-Sm) is highly specific for SLE but has poor sensitivity (sensitivity 24-30%, specificity 96-98%).⁹ The anti-Sm antibody remains positive in patients regardless of disease activity. Therefore, it is useful in diagnosing patients who may be in remission.

Anti-U1RNP is part of the essential diagnostic criteria for mixed connective tissue (MCTD). Hence, it is found in 100% of patients with MCTD. Between 3-69% of SLE patients may have anti-U1RNP.

The anti-Sm and anti-U1 RNP antibodies are detected using ELISA- and flow cytometery-based assays. The antigen preparation in these solid phase assays may be contaminated with other antigens and can cause false positive results.

Anti-SSA/anti-SSB antibodies. Traditionally associated with Sjogren’s syndrome and SLE, anti-SSA may also be found in other autoimmune conditions (RA, primary biliary cirrhosis, polymyositis, etc.). Anti-SSA is directed against two antigens: Ro 52 and Ro 60. Anti-SSB is directed against La antigen. These antibodies are detected using counter immunoelectrophoresis, ELISA, and Western blot.

Anti-SSA antibodies (Anti-Ro antibodies) are present in 50-60% of patients with primary Sjogren’s syndrome, 10-15% of patients with secondary Sjogren’s syndrome, and 35-40% of SLE patients.⁴ Their presence in pregnant women is associated with neonatal lupus and congenital heart block. Anti-SSA antibodies are also found in patients with "ANA negative lupus" and should be checked in patients with high clinical suspicion of SLE who have negative ANA. Anti-SSA antibodies may precede development of SLE. Anti-SSA antibodies are also associated with subacute cutaneous lupus erythematosus (SCLE). Anti-SSB antibodies are present in 40-50% of patients with Sjogren’s syndrome and 15% of patients with SLE. Patients with primary Sjogren’s syndrome in the presence of anti-Ro and anti-La are at greater risk of developing extra glandular manifestations.⁴

Anti-histone antibodies. More popularly associated with drug-induced lupus, the anti-histone antibodies are in fact found in 80% of patients with idiopathic lupus. They are present in more than 95% patients with drug-induced lupus.

Antiribosomal P protein antibodies. The antiribosomal P protein antibodies are specific for neuropsychiatric lupus. These are found in only 10-20% of lupus patients.

Anti-Scl 70, anti-centromere, and anti-U3RNP antibodies. Anti-Scl 70 is found in some patients with systemic sclerosis. It is highly specific (100%) for diffuse systemic sclerosis.¹⁰ The presence of antibody is associated with greater progression to pulmonary disease and widespread skin involvement. Anti-centromere is associated with limited systemic sclerosis. Anti-Scl 70 and anticentromere are mutually exclusive and do not coexist.

Anti-U3RNP is a predictor of pulmonary hypertension and skeletal muscle involvement in scleroderma patients. Anti-PM/Scl autoantibody is associated with overlap syndrome (myositis with scleroderma). Anti-RNA polymerase lll is associated with development of scleroderma renal crisis.

Antibodies in inflammatory myopathies. Myositis-specific antibodies occur in about 30% of patients with dermatomyositis and polymyositis. Three major categories of myositis-specific antibodies are listed in Table 9.

Rheumatoid Factor (RF). RF are autoantibodies that are directed against the Fc portion of IgG molecule. The most common RF used in clinical practice is IgM RF. RF is most commonly used to diagnose RA. A positive RF can be found in rheumatic and non-rheumatic conditions and in healthy subjects (See Table 10).

The specificity of RF in RA is 50-80% and sensitivity is 85-90%. One fifth of RA patients may never develop RF positivity and up to 10% of healthy individuals may have positive RF. Hence, the test is not very useful in confirming or excluding diagnosis of RA. RF factor positivity in a patient with RA is a prognostic marker for more severe,¹² erosive disease and extra articular involvement. RF titer does not correlate with disease activity and serial RF titers are of no value in clinical practice. However, high RF titer (> 1:160) has a higher predictive value for RA.

Anti-cyclic citrullinated petptide antibodies (anti-CCP). Anti-CCP antibodies are directed against the citrulline residues present on proteins after post-translational modification of arginine. They are highly specific for RA (95-98%); however, their sensitivity is only 30-60%. The presence of anti-CCP antibodies helps to confirm diagnosis of RA. They may also be present several years before the development of clinical manifestations of RA. Hepatitis C patients with arthritis may have RF. In such patients, presence of anti-CCP antibodies helps to confirm diagnosis of concomitant RA.

Early, effective treatment of RA may lead to decrease in anti-CCP titers. Anti-CCP positivity is associated with aggressive, erosive disease.¹³ Anti-CCP antibodies may be positive in other diseases including tuberculosis and alpha-1 antitrypsin deficiency. Cigarette smokers with shared epitope have higher incidence of anti-CCP positive RA.

Antineutrophilic cytoplasmic antibodies (ANCA). ANCA testing plays a critical role in diagnosis and classification of various vasculitides. These antibodies stain the cytoplasmic granules in neutrophils. ANCA testing is done using immunoflourescence and ELISA. Immunoflourescence is a screening method that indicates either cytoplasmic or perinuclear staining, causing c-ANCA and p-ANCA pattern, respectively. c-ANCA is classically associated with granulomatosis with polyangiitis (GPA, previously known as Wegener’s granulomatosis) and p-ANCA with microscopic polyangiitis and Churg Strauss syndrome.

ANCA staining is operator dependent¹⁴ and false positives may occur, especially in the presence of positive ANA. A positive test is further confirmed using ELISA, which detects antibodies against specific antigens, namely PR3 and MPO. PR3 is usually associated with c-ANCA and MPO with p-ANCA. The overall ANCA positivity and pattern prevalence in ANCA associated vasculitides is shown in Table 11.

ANCA positivity with the presence of either PR3 or MPO improved positive predictive value for the diagnosis of vasculitis. Biopsy is the gold standard for diagnosis of vasculitis. ANCA and specific antigen positivity should be used only as an adjunct to diagnosis.

Certain drugs (minocycline, hydralazine, propylthiouracil) may be associated with development of ANCA vasculitis. ANCA positivity is seen in various other conditions besides vasculitis (See Table 12). The pattern is usually pANCA or atypical patterns in such cases.

ANCA titers do not correlate with disease activity consistently. Hence, rising ANCA titers alone cannot be used to diagnose relapse.¹⁵ However, ANCA negativity in a vasculitis patient with prior history of positive ANCA may be consistent with remission in some cases.

Antiphospholipid antibodies. Antiphospholipid antibodies are tested when there is suspicion for antiphospholpid syndrome and in patients with SLE. Patients may have history of recurrent arterial or venous thrombi, pregnancy morbidity, and recurrent fetal loss. The presence of biologic false positive serologic test for syphilis may be the first clue to presence of anticardiolpin (aCL), antiß2 glycoprotein1 (antiß2GP1), or lupus anticoagulant.

LAs are antibodies directed against plasma proteins, which also bind phosholipid surfaces. In half of the cases, they may cause prolonged activated partial thromboplastin time (aPTT). When patient’s plasma is mixed with normal plasma, there is no correction of aPTT. This suggests presence of inhibitor since normal plasma would correct any factor deficiency. Thereafter, confirmatory test is performed, usually dilute Russell viper venom time or the hexagonal lipid neutralization test. In the confirmatory tests, the addition of phospholipid corrects the clotting time. LA should not be checked if the patient is on an anticoagulant.¹⁶ In such cases, aCL or antiß2GP1 should be checked.

aCL and antiß2GP1 antibodies are measured by ELISA. The titers may be reported as low, medium, or high. These antibodies should be present in medium-to-high concentration on at least two occasions at least 12 weeks apart to qualify for diagnostic criteria for APS.

Complement

Complement system is part of the innate immune system. Complement deficiency may be inherited (uncommon) or acquired (common). Acquired complement deficiency may occur due to reduced hepatic synthesis (e.g., liver failure) or accelerated consumption by immune complexes (e.g., SLE, cryoglobulinemia, rheumatoid vasculitis, autoimmune hemolytic anemia, autoimmune pancreatitis, membranoprolifertaive glomerulonephritis, and parvovirus infection).

CH50 (total hemolytic complement) assesses classic complement pathway components (C1 to C9). CH50 is low when there is deficiency of any single component. Low C3 and low C4 are present in up to 50% of SLE patients at some point in their course. Low complement levels correlate with lupus nephropathy.¹⁷ However, their use as predictors for lupus flare is controversial.

Inherited deficiency of C1, C2, and C4 may predispose to SLE. Complete C3 deficiency can cause severe recurrent pyogenic infections. Partial C4 deficiency may predispose to SLE. Deficiency of C1 esterase inhibitor leads to low C4 levels and may predispose to SLE.

Cryoglobulins

Cryoglobulins consist of immunoglobulins and complement components that precipitate upon refrigeration of serum. The collection of blood in prewarmed syringe (at 37° C) is imperative to avoid false negative. A cryocrit is determined at 4° C by measuring the packed volume of the precipitate in the wintrobe tube as percentage of total serum collected.

Three types of cryoglobulins are associated with hematologic malignancies, viral infections, and connective tissue disorders (See Table 13). The presence of cryoglobulin does not confer increased morbidity or mortality risk. The prognosis depends on underlying condition. However, severe complications, such as cryoglobulinemic vasculitis or renal failure, may predict poorer outcomes.

Clinical Case Example

An example of how to approach a patient who is believed to have an autoimmune disease might play out like this: A 35-year-old female patient with chest pain on deep breathing and finger joint swelling and pain presents to her primary care physician. After a thorough history and a good physical examination (which revealed a few basilar rales and a 1 cm cervical lymph node), laboratory and x-ray findings may help identify the patient’s problem. Differential diagnoses may include SLE, RA, endocarditis, sarcoidosis, pulmonary embolus (PE) with osteoarthritis, hepatitis C, etc. However, she seems to fit SLE the best. So an ANA is ordered and turns up negative. ANA is a sensitive test but is not very specific, so it is a good test to rule out a diagnosis of SLE if it is negative, since 98% of patients with SLE have a positive ANA. Now there is no need to do a whole battery of tests for SLE, which will save the patient hundreds of dollars. Meanwhile, RF and CCP both were negative, which gave about 80-85% assurance that the patient doesn’t have RA. The hepatitis C screen was negative, as was a VQ scan for PE. A chest x-ray showed some enlarged hilar lymph nodes and early pulmonary fibrosis changes. The lymph node was biopsied and a diagnosis of sarcoidosis was made. The lesson to be learned is to use screening tests first before delving into numerous more specific tests that may not be needed.

Summary

When ordering laboratory tests, a physician needs to have a good reason for doing the test. (See Box above for a description of routine laboratory tests in rheumatology.)The patient’s history should suggest a reason to order a specific test in order to properly interpret the result obtained. In rheumatology, a diagnosis is dependent on the history for about 85% of the information to make that diagnosis. About 10% is dependent on the physical examination. Laboratory and imaging tests make up about 5% of the information for diagnosis. Therefore, it is important to do tests in context with the suspected diagnosis and not go on a "fishing expedition" for lab tests that have little relevance to the patient’s problem. It is very important to know the sensitivity and specificity of a laboratory test in relation to the disease that one is suspecting and the reason for the test one is ordering.

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