LN

Antinuclear Antibodies and Systemic Autoimmune Rheumatic Diseases

Antinuclear Antibodies (ANA’s)

  • Antinuclear antibodies (ANAs) are auto-antibodies specifically directed against nuclear components of human cells.

  • ANAs are primarily associated with autoimmune diseases, particularly those classified as Systemic Autoimmune Rheumatic Diseases (SARDs).

  • ANA testing serves as a screening tool for specific SARDs but is not used for definitive diagnosis.

Systemic Autoimmune Rheumatic Diseases (SARDs)

  • SARDs include the following diseases:

    • Rheumatoid arthritis (RA)

    • Systemic lupus erythematosus (SLE)

    • Sjögren's syndrome (SjS)

    • Systemic sclerosis (SSc)/Scleroderma

    • Other associated conditions

  • The following diseases are notably associated with a positive ANA test:

    • SLE

    • SjS

    • SSc

Autoimmune Diseases: Lupus

  • Lupus is described as a chronic inflammatory autoimmune disease.

  • It can affect various body systems including:

    • Skin

    • Heart

    • Lungs

    • Kidneys

    • Joints

    • Nervous system

  • Types of lupus:

    • Discoid lupus: When only the skin is affected.

    • Systemic lupus erythematosus (SLE): When internal organs are implicated.

  • Demographics:

    • More common in women than in men.

    • Average age of onset is between 20 and 40 years.

SLE Diagnosis

  • When systemic lupus erythematosus (SLE) is suspected, the first diagnostic test is screening for ANAs.

    • Approximately 95% of patients with SLE will have a positive ANA test.

    • This test has low diagnostic specificity since ANAs can be present in other autoimmune diseases, leading to false positives.

  • There are eleven criteria used for diagnosing SLE, of which the presence of 4 or more is strongly indicative of SLE:

    • Malar "butterfly" rash

    • Discoid skin rash

    • Photosensitivity

    • Mucous membrane ulcers

    • Arthritis

    • Pleuritis/pericarditis

    • Kidney abnormalities

    • Neurological disorders

    • Blood count abnormalities

    • Immunologic disorder (anti-DNA or anti-Sm antibodies)

    • Presence of antinuclear antibody.

SLE Diagnosis: Anti-DNA

  • Among the various ANAs, double-stranded DNA (ds-DNA) antibodies are the most specific for SLE.

    • These antibodies are found in approximately 40 to 70 percent of SLE patients.

    • ds-DNA antibodies produce a homogeneous staining pattern in indirect immunofluorescent tests.

SLE Diagnosis: Anti-Sm

  • Anti-Sm (Smith) antibodies are another type of ANA.

    • Around 20 to 40 percent of SLE patients will produce anti-Sm antibodies.

    • These antibodies usually produce a coarsely speckled staining pattern in indirect immunofluorescent testing.

    • Anti-Sm antibodies are highly specific for systemic lupus erythematosus.

Autoimmune Diseases: Sjögren’s Syndrome

  • Sjögren’s syndrome involves the inflammation of glands in the body, particularly those that produce moisture.

    • Inflammation of lacrimal glands can cause eye dryness, irritation, decreased tear production, gritty sensations, infections, and severe corneal abrasions.

    • Inflammation of salivary glands leads to mouth dryness, difficulties in swallowing, dental issues, gum diseases, mouth sores, and swelling.

    • Effect demographic: Mainly affects individuals older than 40, with women being nine times more likely to be affected than men.

Autoimmune Diseases: Scleroderma

  • Scleroderma is characterized as an autoimmune disease of connective tissue.

    • It is identified by the formation of scar tissue (fibrosis) in skin and organs.

    • The affected areas demonstrate thickness and firmness.

    • It can occur in individuals of any age but is most frequently seen in ages 30 to 50.

    • Women are affected four times more often than men.

Antinuclear Antibody Test

  • The test qualitatively detects ANAs in human serum.

    • It employs an indirect immunofluorescence method using human epithelioid (Hep-2) cells grown on slides as the substrate.

    • A fluorescent reagent, specifically fluorescent anti-human gamma globulin, is utilized.

  • Washing Procedures:

    1. Wash after adding the serum sample.

    2. Wash again after adding the fluorescent reagent.

Antinuclear Antibody Test: Components

  • Unknown Components:

    • ANAs present in patient serum samples.

  • Known Components:

    • ANA antigen substrate.

    • Secondary antibody with fluorescent conjugate.

    • Reagent bottles containing known substances.

Limitations of the Test

  1. Some drugs may induce the presence of ANAs.

  2. A positive ANA test does not confirm SLE; it must be correlated with other examination data.

  3. Multiple antibody patterns may arise due to the presence of more than one antibody in the sera.

  4. Abnormal test results may result from allowing the slides to dry before adding the fluorescent conjugate.

Positive Reaction in ANA Testing

  • During a positive reaction:

    • The ANA antigen-substrate complex forms.

    • Positive and negative control slides confirm testing efficacy.

    • The presence of antibodies in serum binds to nuclear DNA within the Hep-2 cells.

    • Bright fluorescence indicates successful binding and presence of ANA.

Protocol for ANA Testing

  • Part 1: Preparation of Samples

    1. Remove a slide from the pouch.

    2. Add 10 microliters of each control (serum samples) to their respective wells (do not spread).

    3. Place the slide in a Petri dish with a moist paper towel underneath to prevent drying.

    4. Incubate for 30 minutes.

  • Part 2: Performing the Test

    1. Remove the slide and rinse with a stream of PBS thoroughly.

    2. Place the rinsed slides in a coplin jar filled with PBS for a 10-minute wash, ensuring agitation.

    3. Drain excess PBS and dip the slide into distilled water to rinse off remaining PBS.

    4. Tap the corner of the slide on a paper towel to remove excess water (avoid blotting or drying).

    5. Return the slide to a moist chamber, flood it with fluorescent reagent, and incubate in darkness for 30 minutes.

    6. Repeat steps 1 and 2 (washing).

    7. Repeat step 3 (tapping to remove water).

    8. Add 2-3 drops of buffered glycerol (mounting medium) to each slide and apply the cover slip gently.

    9. Examine under the fluorescent microscope.

Staining Patterns in ANA Testing

  • Speckled Control:

    • Nuclei appear grainy/speckled.

  • Nucleolar Control:

    • Staining appears clumpy, smooth, or speckled, often high-titered in Scleroderma.

  • Homogeneous Control:

    • Whole nucleus is evenly stained; most typical of systemic lupus erythematosus.

  • SSA/Ro Control:

    • Displays distinctive speckled patterns, with about 10-15% hyper-expressing cells showing strong staining.

  • Negative Control:

    • No discernable pattern is visible.

Review Questions

  • What do ANAs specifically target?

  • What SARDs are associated with a positive ANA test result?

  • Identify the SARD linked to inflammation of moisture-producing glands, the chronic inflammatory condition, and the autoimmune disease of connective tissue.

  • List some criteria strongly indicative of SLE and the number needed for a strong suggestion.

  • Name the antinuclear antibody prevalent in 40 to 70 percent of lupus patients.

  • Identify the extractable nuclear antigens found in 20 to 40 percent of lupus patients.

  • Distinguish between the unknown and known components during testing.

  • Determine how many washes are performed during the protocol and at which points.

  • Describe the different staining patterns and their unique characteristics for quiz preparation.