ANTI-NUCLEAR ANTIBODY TEST

GROUP 6 SANTOS, ROMILDA SARANGAY, JEANNE VYKA SY, ALODY FRANCES TAN, MARIEL LYNNE UMALI, KIMBERLIE UMANDAP JANELLE VERGARA CYRILL

ANA

Anti-nuclear Antibodies

An autoantibody directed against a component of the

nucleus, commonly found in systemic lupus erythematosus (SLE) Screens for presence of other autoimmune defects

Types of ANA
Antibodies to double stranded DNA

Antibodies to histone proteins

Antibodies to Chromatin

Other types of ANA and pattern

Diseases associated with ANA

Systemic lupus erythematosus Sjogren's syndrome rheumatoid arthritis Polymyositis Scleroderma Hashimoto's thyroiditis Addison disease Glomerulonephritis pulmonary fibrosis Cancer Chronic infection

ANA TESTING
I M T E C - A N A T E S T I N G F A N A ( F L O U R E S C E N T A N T I N U C L E A R

ANTIBODY) O T H E R T E S T R E L A T E D T O S L E

Anti-nDNA slide test SLE latex test

IMTEC-ANA Screen

ELISA Quantitative Determination of Antinuclear

Antibodies (Ig(GAM))

Reagents and contents

REAGENTS MTP CAL (white cap) NC (green cap) PC (red cap) Wash (black cap) Dil (blue cap) Con (white cap) SUB- (black cap) Stop (red cap) Adhesive tapes Contents coated with HeLa cell nuclei human serum human serum human serum TRIS buffer Phosphate buffer anti-human-IgGAM conjugate 3,3,5,5-tetramethylbenzidin and hydrogen peroxide Sulfuric acid

Procedure

Principle

Chromogenic substrate with TMB is added, oxidized and produce a blue color Color development is stoped Conjugate binds to previously attached antibody

Specific autoantibodies bind to immobilized autoantigens

Results

Absorbance <40 U/ml 40-55 U/ml >55

Interpretation Negative Equivocal Positive

Additional notes SLE is unlikely Repeat test to verify suggest an autoimmune disease, but further specific testing is required to assist in making a final diagnosis. Falsely elevated in Elders Happens even before symptoms of SLE shows

FANA

Most widely used and accepted test for SLE Extremely sensitive test and relatively easy to

perform, but has low diagnostic specificity Cannot be used to absolutely rule out SLE

Procedure and Principle

Mouse kidney or Human epithelial HEp-2 cells are fixed to slide

react to patient serum.

careful washing to remove unreacted antibodies

an antihuman globulin with a fluorescent tag or an enzyme label such as horseradish is added.

Examine in Microscope

Patterns

- associated with SLE and mixed connective tissue disease -associated with SLE, Sjogren syndrome, scleroderma, polymyositis, rheumatoid arthritis, and mixed connective tissue disease - associated with scleroderma and CREST (Calcinosis, Raynaud's syndrome, Esophogeal dysmotility, Sclerodactyly, Telangiectasia) -associated with scleroderma and polymyositis

Nucleolar pattern

Speckled patter

Homogenous patter

Systemic Lupus Erythematosus (SLE)

Disease most commonly associated with ANA A chronic systemic inflammatory disease marked by

alternating exacerbations and remissions

Associated with inherited deficiencies of complement

components C1q, C2, and C4

Abnormalities of Fc receptors on B cells, macrophages,

dendritic cells, and neutrophils that bind IgG and prevent excess immune reactions

 Uncontrolled autoreactivity of T and B cells leading

to the reduction of autoantibodies Constant presence of antigenic material triggers polyclonal activation of B cells Accompanying alteration in the function of Th1 and Th2 helper cells

Anti-nDNA slide test

Principle
When

serum containing the antibodies combine with latex particles bound with nDNA, agglutination occurs.

Procedure
serum + control - control

Add anti-nDNA latex reagent

Mix with applicator stick

Tilt for 2 mins and observe for macroscopic clumping

Results and Interpretation

Positive Presence of clumping

Negative Smooth suspension The presence of ANA (presence of clumping) in a patient is not diagnostic for SLE. However, having a negative result in this test rules out SLE.

SLE Latex Test

Principle Latex particles, that are coated with deoxyribonucleoprotein, react and form agglutination with the serum of SLE patients due to the presence of antinuclear antibodies.

Procedure and interpretation

Same as Anti-nDNA Slide test

Test is specifically anti-DNP

Test is negative if no difference in agglutination seen between patient serum and negative control. Test is positive if it shows distinct agglutination within 2 minutes as same with the positive control.