Diagnostic tests in rheumatic diseases
Prof Dr Aşkın Ateş
Laboratory tests
• Laboratory results are considered in combination with clinical signs and symptoms and should not replace a thorough history and physical examination in the diagnostic evaluation.
• Measurement of biomarkers can also be useful for monitoring of treatment efficacy and safety as well as for stratification of patients to predict prognosis and treatment response.
• The most common use of blood cell counts and measurements of biochemical markers is to monitor adverse effects of the
various immunosuppressive medications used for the treatment of rheumatic conditions.
White blood cells
• Increased concentrations of neutrophils typically are seen in bacterial infections (e.g., infectious arthritis, septicemia, pneumonia) but also in
active phases of rheumatic diseases such as Rheumatoid arthritis (RA), Adult onset Still disease (AOSD)and vasculitides.
• In patients undergoing immunosuppressive treatment, the presence of neutropenia should always raise suspicion of drug-related bone marrow suppression.
• In contrast, neutropenia in association with splenomegaly is a characteristic feature of Felty syndrome.
• Whereas lymphocytosis is present during several viral infections, leukopenia and lymphopenia are commonly seen in active phases of Systemic lupus
erythemathosus(SLE) as well as with immunosuppressive drug treatment.
Platelets
• Thrombocytosis can accompany active phases of autoimmune diseases
such as RA and vasculitis, but thrombocytopenia can be related to the presence of antithrombocyte antibodies, for example, in active SLE.
• Furthermore, decreased thrombocyte counts raise suspicion of drug-induced toxicity.
Red blood cells
• Anemia in rheumatic diseases most commonly reflects decreased production of red blood cells in the bone marrow caused by continued inflammation, with increased hepcidin production leading to disturbed iron metabolism.
• Anemia of chronic disease is commonly normocytic and normochromic;
however, microcytic hypochromic anemia also can be associated with chronic disease.
• Anemia caused by chronic or acute gastrointestinal bleeding is also a frequent adverse effect of nonsteroidal anti-inflammatory drugs (NSAIDs), with
glucocorticosteroids contributing to an increased risk
• Hemolytic anemia occurs in active SLE with detection of antierythrocyte antibodies and in other immune complex mediated conditions.
• Pernicious Anemia is a condition in which the body cannot produce enough red blood cells due to a lack of vitamin B12.
People with pernicious anemia cannot absorb enough B12 from food because of a lack of intrinsic factor--a protein made in the stomach--which leads to vitamin B12 deficiency. Pernicious
anemia is frequently associated with autoimmune rheumatic diseases especially SLE, Sjögren’s syndrome and RA.
Kidney function tests And Urinalysis
• Kidney function tests are routinely performed before and after initiation of treatment with antirheumatic drugs, including NSAIDs, to monitor for adverse effects. For this purpose, serum creatinine levels or estimated glomerular filtration rate (eGFR) provides sufficient information.
• Connective tissue diseases (CTDs) and systemic vasculitides are frequently associated with kidney involvement, causing glomerular and interstitial nephritis. In cases of suspected nephritic or nephrotic syndrome, urine tests and kidney biopsy are standard procedures.
• Due to glomerulonephritis nephrotic or nephritis range of proteinuria occurs in patients with CTDs (especially SLE) and vasculitis.
Kidney function tests And Urinalysis
• Urinalysis is also useful for monitoring of kidney involvement and should include detection and quantification of proteins as well as of hematuria and leukocyturia.
• Patients with persistent proteinuria should undergo a quantitative measurement of total protein excretion.
Quantification methods include the ratio of protein to creatinine concentration in a random, spot urine collection and the 24-hour urine protein collection.
• The gold standard for measurement of protein excretion is a 24- hour urine collection
SYSTEMIC LUPUS ERYTHEMATOSUS AND RENAL İNVOLVEMENT
• Kidney involvement with SLE, lupus nephritis (LN), is common and is seen in up to 50% of those with SLE. Lupus nephritis is an immune complex
glomerulonephritis.
• Regular screening for renal disease including measuring serum creatinine and monitoring for proteinuria is important on a regular ongoing basis for these patients.
• LN portends significant morbidity and mortality.
• Renal biopsy is the gold standard for diagnosis.
• In clinical practice, the elevated anti-dsDNA antibody titer concomitant with reduced complement C3 and C4 levels has become the predictive and disease-activity surrogate biomarkers in LN.
• Concerns for lupus nephritis are raised when there is greater than 0.5g of protein in a 24 hour urine collection or greater than 3+ protein on a urine dipstick.
• Often times there will be also be concomitant hematuria (>5RBC or WBC/
high power field) and casts on urinalysis (UA).
Synovial fluid analysis
• Synovial fluid analysis is helpful for
determining the underlying cause of arthritis, particularly for septic or crystal-induced
arthritis.
• The white cell count, differential count,
cultures, Gram stain, and crystal search using polarized light microscopy are the most useful studies.
• Results of synovial fluid analysis can be used to categorize the fluid as non- inflammatory, inflammatory, septic, or hemorrhagic based upon the clinical and laboratory analysis.
Noninflammatory synovial fluid
Examples of conditions associated with noninflammatory synovial fluid include osteoarthritis, avascular necrosis, or a meniscal tear.
Inflammatory synovial fluid
A wide range of conditions are associated with inflammatory synovial fluid such
as rheumatoid arthritis, crystal-induced arthritis (eg, gout, pseudogout), or spondyloarthritis.
Septic synovial fluid
Septic effusions may be due to bacteria, mycobacteria (tuberculosis), or fungus.
Hemorrhagic synovial fluid
Large numbers of red blood cells give synovial fluid the appearance of being hemorrhagic.
They may be due to hemophilia, anticoagulation or other hemorrhagic diathesis, trauma (with or without fracture), neuropathic arthropathy, and tumor (including pigmented
villonodular synovitis, synovioma, hemangioma, and other benign or malignant neoplasms)
Crystal arthritis
• Patients suspected of calcium pyrophosphate dihydrate (CPPD) disease or gout should undergo arthrocentesis and synovial fluid analysis of the affected joint must be done.
• The reference standard for the diagnosis of CPPD and gout are based on the identification of CPP crystals or Monosodium urate crystals (MSU)in synovial fluid by compensated
polarized light microscopy.
Uric acid and Gout
• Uric acid measurement is commonly included in the workup of patients with arthritis, and levels are elevated in 90% of patients with gout.
• Gout is a disease resulting from the deposition of monosodium urate crystals (MSU) in synovial fluid and other tissues or the formation of uric acid stones in the kidney.
• Although the prevalence of gout is equal in men and women, men are six times more likely to have serum uric acid
concentrations above 7 mg per dL.
• Yet healthy people can have elevated levels as well, and the definitive diagnosis of gout depends on demonstration of uric acid crystals (MSU) in synovial fluid.
Uric acid crystals in synovial fluid
Several classes of crystalline material are found in the joints,
Monosodium urate monohydrate (MSU) crystals are needle-shaped, 5 to 30 μm in length, and highly birefringent.
They can be distinguished from other crystals by their properties in polarized light with an interference plate in the system.
Urate crystals appear either yellow or blue
depending on the direction of their long axis of the polarizer; when the long axis of the polarizer is parallel to the long axis of the MSU crystal, the crystal will be yellow.
Other crystals (e.g., calcium pyrophosphate
dihydrate [CPPD] crystals) are also yellow and blue, but the direction of the long axis of yellow- and blue- appearing crystals is reversed. CPP crystal showing weakly positive birefringence in compensated polarized light microscopy.
Note that the CPPD crystals appear duller and that the apparent color of the crystal is the opposite of the urate crystals when related to the long axis of the crystal.
Calcium pyrophosphate dihydrate crystal deposition disease (Pseudogout)
• Calcium pyrophosphate dihydrate crystal deposition disease (CPPD) is an inflammatory arthritis produced by the deposition of calcium
pyrophosphate (CPP) crystals in the synovium and periarticular soft tissues.
• It is the third most common inflammatory arthritis. Diagnosis is suspected on the basis of the clinical picture and radiographic/laboratory findings.
• The reference standard for the diagnosis of CPPD is based on the
identification of CPP crystals in synovial fluid by compensated polarized light microscopy.
Acute-phase reactants
• The acute-phase response occurs in a wide variety of inflammatory conditions, including various infections, malignancies and inflammatory rheumatic disorders.
• Currently, the most widely used laboratory tests to monitor
inflammation are the erythrocyte sedimentation rate (ESR) and C- reactive protein (CRP) level.
• Of note, ESR levels are usually physiologically higher in women and increase with age and during pregnancy.
• CRP levels in particular have been reported to be higher among obese patients.
• Therefore ESR and CRP are non-specific acute phase reactants for inflammatory rheumatic diseases.
• The acute-phase proteins are mainly produced by hepatocytes upon stimulation by cytokines (e.g., interleukin-6, interleukin-1, and tumor necrosis factor).
• The most important acute-phase proteins whose levels increase during inflammation (positive reactants) are CRP, fibrinogen,
S100 proteins, ferritin, serum amyloid protein A, and several complement components, especially C3.
• In inflammatory conditions, increased plasma levels of fibrinogen and immunoglobulins as well as a frequently associated anemia can increase the ESR.
• Serial measurements of ESR and CRP are valuable for monitoring the level of inflammation in patients with inflammatory
rheumatic diseases such as RA.
SEROLOGIC TESTING AUTOANTIBODIES
Rheumatoid factor
• Rheumatoid factor (RF) is the traditional
designation for autoantibodies directed at the Fcγ chains of IgG molecules.
• The most commonly measured autoantibody is the IgM isotype.
• In clinical practice, laboratories tend to test only for IgM RF, but RF can belong to all major
immunoglobulin classes (IgG, IgA, IgM).
Rheumatoid factor
• The main indications for RF testing in diagnosis are suspicion of RA or Sjögren syndrome. The sensitivity of RF for RA ranges from 70% to 80%.
• RF also commonly appears in mixed connective tissue disease, SLE, cryoglobulinemia.
• It is thus apparent that the specificity of RF in differentiating RA from some other rheumatologic conditions is poor.
However, with higher titers of RF, the specificity increases, and RF testing becomes a more useful laboratory tool for the diagnosis of RA.
Rheumatoid factor
• Increased polyclonal immunoglobulin production in diverse nonrheumatic diseases can cause a positive RF.
• This phenomenon can be seen in primary biliary cirrhosis, sarcoidosis, malignancies, and infections such as hepatitis B and C, tuberculosis, infective endocarditis.
• An increased prevalence of RF is also found in smokers and in the healthy elderly population.
Anti-citrullinated protein antibodies (ACPAs)
• ACPAs are autoantibodies (antibodies to an individual’s own proteins) that are directed against peptides and proteins that are citrullinated.
• ACPAs recognize peptides and proteins containing citrulline, a non-standard amino acid generated by the posttranslational modification of arginine
by peptidylarginine deiminase enzymes, in a calcium-dependent process known as citrullination.
• They are present in the majority of patients with rheumatoid arthritis.
Clinically, cyclic citrullinated peptides (CCP) are frequently used to detect
these antibodies (ACPAs) in patient serum or plasma (then referred to as anti–
citrullinated peptide antibodies).
• ACPAs are detected in approximately 2/3 of RA patients with a diagnostic specificity of 98%.
Anti-cyclic citrullinated
antibody (Anti-CCP antibody) ELISA
• The used the 2nd generation anti-CCP (anti-CCP2) enzyme-linked immunosorbent test (ELISA) allowed the widespread routine
testing for antibodies directed against citrullinated epitopes (peptides) as a biomarker for RA .
• Sensitivity recently increased to nearly 80% after we used the 2nd generation anti-CCP enzyme-linked immunosorbent test (ELISA).
• The high specificity (98%) of anti-CCP in patients with RA can exclude other rheumatic or immune diseases in patients with positive anti-CCP.
• The diagnostic performance of anti-CCP is superior to RF because they can be detected with a high specificity and moderate
sensitivity for RA using citrullinated proteins or peptides in ELISA.
Antinuclear antibodies
• ANA testing serves as a screening tool for connective tissue diseases.
• However, ANAs are seen in individuals with many
rheumatologic conditions as well as in many healthy people.
• The gold standard for ANA screening is indirect
immunofluorescence (IIF) on human epithelial (HEp-2) cells.
• Subsequently, differentiation of a positive ANA result should be performed using solid-phase immunoassays such as ELISA.
ANA testing
• The presence of ANAs is a serologic hallmark of connective tissue disorders such as SLE, Sjögren syndrome, mixed
connective tissue disease, systemic sclerosis, and
undifferentiated connective tissue disease, as well as of autoimmune hepatitis.
• However, finding antibodies by itself does not signify disease because healthy people (especially family members of patients with connective tissue diseases, older adults, and patients using drugs such as sulfasalazine and isoniazid) can show clinically
irrelevant positive ANA reactivity.
• A high titer increases the likelihood that the presence of antibodies is related to an autoimmune disease.
Titers less than 1/40 should be considered negative (20-30% of healthy people)
Titers of 1/40 to 1/160 are positive at low titer
(further workup is not recommended in the absence of specific symptoms)
Titers equal to or greater than 1/160 are positive.
The percentages of ANA titers in healthy peoples
ANA nucleolar staining
• Anti-dsDNA antibody testing is very specific (95%) but not as sensitive (70%) for SLE, which makes the test result part of the classification criteria for SLE.
• Anti-dsDNA antibodies also might have a pathogenic role in lupus nephritis and are commonly associated with disease activity. The usual picture in active lupus nephritis is increased anti-dsDNA antibody along
with decreased C3 and C4, with levels of all tending to normalize with control of active kidney disease.
Anti–Ro (SS-A) and anti–La (SS-B) antibodies
• Anti–Ro and anti–La antibodies are part of the classification criteria for Sjögren syndrome but also are frequently detectable in SLE patients.
• They have been associated with subacute cutaneous lupus and
photosensitivity. Anti–Ro and anti–La antibodies have also been associated with neonatal lupus and congenital heart block because they cross the
placental barrier.
• Therefore, during pregnancy, the fetus should be screened for congenital heart block by ultrasonography, and newborns of anti-Ro antibody-positive mothers should avoid sunlight exposure during the first weeks of life.
• These antibodies produce a fine speckled pattern in ANA IIF.
Anticentromere and anti–Scl-70 antibodies
• Anticentromere and anti–Scl-70 autoantibodies are the specific and most frequent markers for systemic sclerosis, each
detectable in up to 40% of patients. Anticentromere antibodies produce a typical pattern in ANA IIF by staining the centromere region of chromosomes.
• The target of anti–Scl-70 is topoisomerase I, and it shows a nucleolar staining.
• Anticentromere antibodies are more frequently observed in the context of localized scleroderma and pulmonary hypertension.
• Patients who test positive for anti–Scl-70 typically have diffuse scleroderma and pulmonary parenchymal involvement.
Antineutrophil cytoplasmic antibodies (ANCAs)
• Antineutrophil cytoplasmic antibodies (ANCAs) represent a subgroup of neutrophil-specific
autoantibodies. They are commonly directed to the azurophil granule proteins myeloperoxidase (MPO) and proteinase 3 (PR3).
• ANCAs are characteristic of patients with
necrotizing vasculitides such as granulomatosis with polyangiitis, microscopic polyangiitis, and eosinophilic granulomatosis with polyangiitis.
Antineutrophil cytoplasmic antibodies (ANCAs)
• In IIF assays, ANCA directed at PR3 typically gives rise to a coarse granular fluorescence pattern in the cytoplasm of neutrophils and monocytes using ethanol-fixed leukocytes and is therefore called cytoplasmic ANCA (c-ANCA).
• In contrast, MPO-ANCA produces a perinuclear staining pattern on neutrophils and monocytes (p-ANCA).
• A positive result on an IIF screening test for p-ANCA or c- ANCA should be confirmed using a specific ELISA using MPO (for p-ANCA) and PR3 (for c-ANCA) as antigens, respectively.
Cytoplasmic staining pattern of antineutrophil cytoplasmic antibodies (c-ANCA)
Perinuclear staining pattern of antineutrophil cytoplasmic antibodies (p-ANCA)
