Clinical Immunology & Serology - Autoimmunity Part 2

Clinical Immunology & Serology

Chapter 15: Autoimmunity Part 2

Systemic Lupus Erythematosus (SLE)

  • Definition: A chronic systemic inflammatory disease that affects multiple organ systems.

  • Epidemiology:

    • Higher prevalence in females over males at a ratio of 9:1.

    • More common in African Americans and Hispanics than in Caucasians.

    • Typically diagnosed between the ages of 20 and 40 years.

  • Prognosis:

    • The 5-year survival rate is approximately 95%.

  • Etiology:

    • Development appears to be influenced by both genetic and environmental factors.

    • Environmental triggers include:

    • Exposure to UV light.

    • Certain medications (causing transient drug-induced lupus).

    • Possibly infectious agents.

    • Over 60 genetic loci associated with lupus.

  • Autoantibody Production:

    • Patients develop numerous autoantibodies (over 100 identified), such as:

    • Anti-double-stranded DNA (anti-dsDNA).

    • Autoantibodies directed against nuclear components, lymphocytes, RBCs, etc.

    • Immune complexes formed trigger complement activation, neutrophil chemotaxis, and inflammation.

Pathophysiology of SLE

  • Autoantibody Dynamics:

    • Presence of autoantibodies can precede clinical symptoms by several years.

    • Most patients have multiple circulating autoantibodies.

  • Mechanisms of Autoantibody Formation:

    • Abnormal apoptosis and ineffective clearance of apoptotic bodies may expose cellular components, leading to autoantibody formation.

    • Anti-dsDNA antibodies occur in 70% of SLE patients and are highly specific for the disease.

    • IgG-dsDNA complexes are significant pathologically, as their moderate size allows them to lodge in the glomerular basement membrane.

Clinical Manifestations of SLE

  • Initial Symptoms:

    • Fatigue, weight loss, fever, anorexia, and malaise often emerge at the onset.

  • Common Clinical Features:

    • Joint involvement.

    • Skin rashes.

    • Renal involvement.

    • Cardiac involvement.

    • Neurological symptoms.

    • Hematological manifestations such as anemia, leukopenia, and thrombocytopenia.

  • Disease Course:

    • Characterized by phases of remission and relapsing flares.

    • Drug-induced lupus is typically milder and resolves once the offending drug is discontinued.

  • Treatment Modalities:

    • Treatment options include immunomodulating drugs, immunosuppressive drugs, and monoclonal antibodies targeting B cells.

Laboratory Diagnosis of SLE

  • Essential Laboratory Tests:

    • Complete blood count (CBC).

    • Urinalysis and creatinine levels to assess kidney function.

    • C-reactive protein (CRP) and erythrocyte sedimentation rate (ESR) – both elevated during flares.

    • Antinuclear antibody (ANA) testing:

    • Initial screening test; if positive, further testing is required.

    • Specific tests for anti-dsDNA and anti-Smith (anti-Sm) antibodies known for their high specificity for SLE.

    • Levels of anti-dsDNA will typically increase before a disease flare.

    • Complement quantification (C3 and C4 levels may be decreased during flare due to consumption).

    • Additional tests ordered based on specific patient symptoms.

Antinuclear Antibodies (ANAs)

  • ANA Overview:

    • Directed against antigens located within cell nuclei.

    • Present in more than 95% of lupus patients.

  • Types of ANAs:

    • Anti-ds DNA (specific for lupus).

    • Anti-ss DNA.

    • Anti-histones and nucleosomes (ab to histones H2A, H2B, and ssDNA - associated with drug-induced lupus).

    • Antibodies targeting centromeres or nucleolar components.

    • Anti-extractable nuclear antigens (e.g., anti-Sm, anti-RNP, anti-SS-A, anti-SS-B).

  • Relevance in Other Conditions:

    • ANAs can also be present in several other autoimmune diseases, chronic infections, cancers, pregnancy, and are found in 5% of healthy individuals and up to 30% in the elderly.

Fluorescent ANA (FANA) Test Principle

  • Testing Method:

    • Indirect immunofluorescence (IIF) is the widely used method for ANA testing:

    • Highly sensitive, can detect many antibodies, inexpensive, and easy to perform.

    • Antigens maintained in original form allow localization identification.

    • Useful for screening in symptomatic patients and guides subsequent testing.

    • Can determine antibody titers.

  • Procedure:

    • Incubate patient serum with HEp-2 cells fixed onto a microscope slide.

    • Wash and then incubate with fluorescein-labeled anti-human IgG.

    • Wash slides and view under a fluorescent microscope.

  • Limitations:

    • Process is time-consuming, requires technical skill, and results can be subjective.

Alternative ANA Testing Options

  • Enzyme-linked immunosorbent assay (ELISA).

  • Chemiluminescence.

  • Microsphere multiplex immunoassays.

  • Ouchterlony double diffusion.

Immunofluorescence Testing Using Crithidia luciliae

  • Testing Overview:

    • An indirect immunofluorescence (IIF) test using Crithidia luciliae as a substrate.

    • C. luciliae contains a circular organelle known as a kinetoplast, which is rich in dsDNA.

  • Purpose:

    • Employed to detect antibodies to dsDNA and assess anti-dsDNA antibody titers; free of other human nuclear antigens and ssDNA contamination.

Anti-Phospholipid Antibodies

  • Characteristics:

    • Bind to phospholipids and phospholipids complexed with protein.

    • Found in approximately 60% of lupus patients.

  • Clinical Implications:

    • Associated with increased risk of deep vein and arterial thrombosis.

    • Linked to recurrent pregnancy loss, potentially causing false-positive results in nontreponemal tests for syphilis and lupus anticoagulant assay.

    • Example: lupus anticoagulant can lead to prolonged activated partial thromboplastin time (APTT) and prothrombin time (PT) not corrected with mixing studies, heightening risks of clotting and miscarriage.

  • Testing Methodology:

    • Testing can be performed using noncompetitive ELISA.

Rheumatoid Arthritis (RA)

  • Definition: Chronic, symmetric, erosive arthritis of peripheral joints progressing potentially to joint deformity and disability.

  • Epidemiology:

    • Generally diagnosed between ages 25 and 55.

    • Overall incidence shows a 3:1 female to male ratio, with the highest prevalence in women over age 65.

    • Approximately 25% of patients may also develop osteoporosis.

    • Some can present with:

    • Subcutaneous nodules.

    • Pericarditis.

    • Interstitial lung disease.

    • Vasculitis.

  • Prognosis: Advances in treatment offer better quality of life and outcomes due to earlier detection and intervention.

Etiology of RA

  • Genetic Associations:

    • RA has links to more than 100 genetic regions.

    • Positive tests for Rheumatoid Factor (RF) or anti-cyclic citrullinated peptide antibody (anti-CCP) are common.

    • Epigenetic modifications, including DNA methylation, may contribute to disease predisposition.

    • Environmental risk factors such as smoking increase the risk of RA by a factor of two.

Pathology and Treatment of RA

  • Joint Pathology:

    • Inflammatory processes lead to bone and cartilage destruction.

    • Observed lesions in affected joints exhibit increased cell lining of the synovial membrane, leading to a structure referred to as pannus.

    • This hyperplastic growth invades cartilage and joint space; inflammatory immune cell infiltrate is characteristic.

  • Cytokine Role in RA:

    • Pro-inflammatory cytokines are elevated, notably tumor necrosis factor alpha (TNF-a), which stimulates further cytokine production and acts as a chemoattractant. Other significant cytokines include IL-1, IL-6, and IL-17.

    • Cytokines induce matrix metalloproteinases (MMPs) from fibroblasts and macrophages, resulting in cartilage degradation.

    • Overactive osteoclasts erode bone while TNF-a inhibits bone formation, contributing to localized and systemic osteoporosis.

Autoantibodies in RA

  • Immune Complexes:

    • Autoantibodies combine with antigens to form immune complexes within the joints.

    • Production of autoantibodies may escalate as cytokine levels rise.

  • Types of Autoantibodies:

    • Rheumatoid factor (RF): Reacts with the Fc portion of IgG and is found in approximately 80% of RA patients but is not specific for the condition.

    • RF may enhance macrophage activity and antigen presentation to T cells.

    • Anti-CCP antibodies: Highly specific for RA and react to cyclic citrullinated peptide. The pathway of citrullination is due to certain enzymes when granulocytes and macrophages die.

    • C1 complements bind to immune complexes and stimulate the classical complement pathway resulting in further joint inflammation and damage.

Clinical Signs and Symptoms of RA

  • General Symptoms:

    • Non-specific initial symptoms such as malaise, fatigue, fever, weight loss, and transient joint pain primarily affecting small joints of hands and feet, presenting symmetrically.

    • Morning joint pain and stiffness that improves throughout the day.

    • Progression to involvement of larger joints.

  • Systemic Complications:

    • Ongoing inflammation necessitates therapy to avert permanent joint damage and deformities.

    • Some individuals may develop nodules over bones and other organs (e.g., lungs, spleen).

    • About 10% may also experience Sjögren's syndrome as a comorbidity.

    • A major cause of mortality associated with RA is cardiovascular disease, particularly arteriosclerosis.

  • Treatment Approaches:

    • Nonsteroidal anti-inflammatory drugs (NSAIDs; e.g., aspirin, ibuprofen) are no longer the first line of treatment.

    • Disease-modifying anti-rheumatic drugs (DMARDs), predominantly methotrexate, are standard treatment.

    • Glucocorticoids, such as prednisone, can be included for treatment plans.

    • Biologic agents specifically targeting TNF-a are also utilized in management.

Laboratory Testing for RA

  • Rheumatoid Factor Testing:

    • 70-90% of RA patients test positive for RF, though may also appear in 5% of healthy individuals and 10-25% in individuals over age 65 and in patients with other autoimmune diseases.

    • Latex agglutination tests specifically check for IgM.

    • More advanced assays like ELISA are prevalent for testing IgG and IgA RF.

    • Anti-CCP Testing:

    • 95% of RA patients demonstrate positivity for anti-CCP antibodies.

    • ELISA assays are utilized as anti-CCP can predict disease onset years before it becomes clinically evident.

    • Anti-CCP positivity correlates with poorer prognostic outcomes in RA patients.

    • Combined testing for RF and anti-CCP yields a specificity range of 98-100%.

  • Additional Diagnostics:

    • Approximately 40% of RA patients may show positive ANA with low titers and sometimes a speckled pattern against RNP (a small nuclear RNA).

    • Inflammatory markers like ESR and CRP are elevated during active disease, and complement levels may appear normal to elevated in chronic conditions.