Resident Editor: Megan Lockwood, M.D.

Faculty Editor: Jonathan Graf, M.D.

Background

• Basic approach prior tordering any rheumatologic lab:
  • What autoimmune condition are you testing for?
  • What is the pre-test probability of the patient having the disease your testing for?
  • What are the limitations of the test you are ordering?

Nonspecific Inflammatory Markers

• Erythrocyte Sedimentation Rate (ESR)
  • Measures the distance fallen by erythrocytes in a test tube over one hour
    • Elevated fibrinogen likely causes RBCs tsediment at a faster rate
  • A nonspecific, indirect marker of inflammation that can be elevated in infectious, autoimmune, or neoplastic conditions
    • Causes of elevated ESR (think causes of elevated fibrinogen/RBC’s): anemia, hypoalbuminemia, ESRD, DM, pregnancy, any state with presence of acute phase reactants
    • Causes of decreased ESR (think diminished fibrinogen/RBC’s): polycythemia, CHF, cryoglobulinemia, hypofibrinogenemia
  • Does not have specific diagnostic utility for rheumatic diseases, with the exception of polymyalgia rheumatica and giant cell arteritis
  • Some clinical utility in monitoring disease activity or response ttherapy in rheumatoid arthritis, temporal arteritis, and osteomyelitis
  • ESR rises with age:
    • Tcalculate the upper limit of normal: ESR ≤ age/2 (+5 in women)
• C-reactive protein (CRP)
  • An acute phase reactant produced in the liver
  • More dynamic than ESR and tracks inflammatory event more closely in time
    • Increases in 4-6 hrs and normalizes within 1 week
  • Causes of elevated CRP: inflammatory conditions, obesity (adipose tissue makes iL-6 which can elevate CRP but rarely over 10 mg/L)
  • Not affected by anemia, gender, pregnancy, renal failure, (unlike ESR)
• Complements
  • Classic complement cascade is activated by immune complexes
    • Integrity of entire classical cascade measured with CH50 functional assay
    • C3/C4 are most commonly measured individual components of pathway
  • Decreased level with increased complex-medicated activity (SLE, cryoglobulinemia)
  • Increased levels with other inflammatory disorders as they are acute phase reactants
  • C4>>C3 may indicate cryoglobulins
  • Other causes of low complements: endocarditis, post-strep glomerulonephritis and inherited deficiencies

Autoantibodies

• Anti-Nuclear Antibody (ANA) – most commonly and accurately tested by indirect immunofluorescence
  • Auto-antibodies directed against intra-nuclear antigens (the test is NOT for the specific antigen, just detects the presence of these antibodies)
    • Positive ANA: can then use more specific testing (the “sub-serologies”) tlook for the specific antigen
      • Testing the sub-serologies can help tnarrow the diagnosis and determine the clinical significance of the positive ANA
        • Examples: anti-dsDNA, anti-histone, anti-Smith, anti-RNP, anti-SSA, anti-SSB, anti-RNP, anti-centromere, SCL-70
      • Once positive, ANA does not need tbe rechecked; fluctuating titers have nclinical significance
      • Titers ≥ 1:160: more likely trepresent true-positive results
      • Titers ≤ 1:40: less specific and less likely trepresent true rheumatologic diseases
      • Staining pattern has clinical significance (gives a clue as twhich molecules are being recognized by the antibodies): nuclear, cell cycle-associated, cytoplasmic
    • Negative ANA: generally nindication ttest for other antibodies
      • Exception: test for anti-SSA which on older assays for ANA could be missed. Test when there is high suspicion for Sjögren’s Syndrome or other disease accociated with SSA positivity.
  • Significance:
    • Nearly 100% sensitive for SLE (but remember, only one part of the criteria for SLE)
    • 30% of healthy people have positive ANA (highly non-specific, see below)
  • Common non-lupus related causes of a positive ANA:
    • Demographics: elderly, pregnant women, family history of SLE
    • Drug-induced:hydralazine, procainamide, quinidine, isoniazid, minocycline (homogeneous pattern reflects antibodies thistones)
      • Absence of anti-histone antibodies effectively rules out the disease (very high sensitivity, low specificity)
    • Other rheumatologic diseases: RA, scleroderma, Sjögren’s, MCTD, polymyositis, derematomyositis
    • Other autoimmune diseases: autoimmune thyroiditis, type 1 DM, pulmonary fibrosis, multiple sclerosis
    • Non-autoimmune diseases: HIV and other viral infections, liver disease, malignancy, endocarditis
• Anti-RNP (ANA sub-serology)
  • 100% sensitivity for mixed-connective tissue disease (diagnostic criterion)
  • Alscommon in SLE
• Anti-SSA (Ro) and Anti-SSB (La) (ANA sub-serology)
  • High sensitivity for primary Sjögren’ssyndrome
  • Dual antibody patients more likely thave sjogren’s syndrome with more severe and extra-glandular disease
  • Much lower percentage of positive antibodies in secondary Sjogren’s syndrome
  • Can alssee in SLE, especially with cutaneous involvement, and neonatal lupus
• Anti-centromere (ANA sub-serology)
  • Classically associated with limited scleroderma (lcSSc) “CREST” syndrome
• Rheumatoid Factor (RF)
  • An IgM (less commonly IgG and IgA) antibody directed against the Fc portion of self-IgG.
  • 80% sensitive for RA. Less specific for RA:
    • Commonly positive in many other conditions (see below)
    • All RF-positive arthritis is not RA!
  • Up t40% of RA patients may be RF negative early in their disease course, but many seroconvert over time.
  • Rf positivity correlates with disease severity (erosive disease) and extra-articular manifestations of RA
    • Once positive, serial RF titers are not useful for following disease progression
  • Common causes of a positive RF other than RA:
    • Demographics: elderly
    • Other autoimmune diseases: Sjogren’s, cryoglobulinemia, SLE, systemic sclerosis, mixed connective tissue disease
    • Non-autoimmune diseases: Chronic infections (HCV, endocarditis, osteomyelitis, tuberculosis, HIV), liver disease, sarcoidosis, multiple myeloma, MGUS.
• Anti-citrullinated peptide (anti-CCP)
  • Antibodies against proteins containing citrulline, an aminacid created via a post-translational modification of arginine and
  • The immune response tcitrullinated proteins is thought tbe key tthe pathogenesis of RA
    • Newer generation of tests with similar sensitivity (80%) and improved specificity (95%) for RA compared tRF (should be ordered simultaneously in the appropriate clinical setting)
    • Can be detected years before onset of clinical disease
    • Associated with more aggressive and erosive disease
• Cryoglobulins
  • Immunoglobulins that reversibly precipitate at cold temperatures
    • Precipitation can lead tvasculitis (as they often bind with proteins and form immune complexes)
  • Types of cryoglobulins (Brouet classification):
    • Type I: monoclonal Ig (often IgM or IgG isotypes)
      • Usually result from monoclonal expansion of malignant clone
        • Malignant: multiple myeloma, Waldenström’s, lymphoma
        • Indolent: MGUS
        • Secondary tlymphoproliferative disorder: CLL
    • Type II: monoclonal Ig + polyclonal Ig (mixed)
      • RF activity of monoclonal cryoglobulin à binding of polyclonal IgG tFc
        • Immune complex formation: Activates complement, associated with small and medium-sized vessel vasculitis
      • Often secondary tchronic infections: HBV, HCV, bacterial, parasitic
      • Seen in other autoimmune conditions: Sjögren’s, SLE, RA
    • Types III: polyclonal Ig (mixed)
      • RF activity of polyclonal cryoglobulin à binding of polyclonal IgG tFc
        • Immune complex formation: Activates complement, associated with small and medium-sized vessel vasculitis
      • As Often secondary tchronic infections: HBV, HCV, bacterial, parasitic
      • Seen in other autoimmune conditions: Sjogren’s, SLE, RA
    • Anti-neutrophil cytoplasmic antibody (ANCA)
      • Antibodies against cytoplasmic granules of neutrophils
        • Similar tANA, except against neutrophil-specific antigen
      • Immunofluorescence patterns:
        • C-ANCA
          • Correlates with serine protease-3 (PR-3) cytoplasmic protein
          • Associated with GPA (>90% sensitivity, specificity)
        • P-ANCA
          • Correlates with myeloperoxidase (MPO) peri-nuclear protein
          • Associated with MPA, EGPA, renal-limited vasculitis
        • Atypical p-ANCA pattern
          • May be seen in other autoimmune diseases with or without vasculitis (i.e. IBD, PSC, autoimmune hepatitis, connective tissue diseases)
          • Alsseen in drug-induced forms of vasculitis
      • Extreme caution with interpretation:
        • • IF results must be confirmed by ELISAs for PR-3 and MPO
          • Pre-test probability is key when testing for rare diseases such as vasculitis, and positive tests should be interpreted with caution by experts
          • Titers may normalize with treatment and when disease is quiescent (beware the false negative in the setting of high pre-test probability!)
      • High titer atypical P-ANCA positivity associated with Levamisole-induced vasculopathy/vasculitis

References

Yazdany J, Schmajuk G, Robbins M, et al. Choosing wisely: the American College of Rheumatology’s Top 5 list of things physicians and patients should question. Arthritis Care Res. 2013;65(3):329-339.

Kolopp-Sarda MN, Miossec P. Cryoglobulins: An update on detection, mechanisms and clinical contribution. Autoimmun Rev. Epub ahead of print: 2018 Mar 8.

SeP, Stone JH. The antineutrophil cytoplasmic antibody-associated vasculitides. Am J Med. 2004;117(1):39-50.

Graf J. Interpreting Rheumatologic Lab Tests. 41^st Annual Advances in Internal Medicine in San Francisco, CA, May 2013. Oral Presentation.