Indirect immunofluorescence

what is indirect immunofluorescence?

Indirect immunofluorescence, or secondary immunofluorescence, is a technique used in laboratories to detect circulation. autoantibodies in patient serum. It is used to diagnose autoimmune Blistering diseases.

• Unlabeled primary antibodies (used as a marker for Cancer, diabetes, and Alzheimer's disease) of the patient's serum bind to the target molecule in previously prepared tissue samples.
• Secondary antibodies (used as a marker for HIV), conjugate with a fluorescent dye such as fluorescein isothiocyanate (FITC), binds to the primary antibody.
• When exposed to light, the fluorescent dye becomes excited and emits a wavelength that can be seen under a fluorescent microscope.

The tissue substrates used (either monkey esophagus or human skin) will depend on the suspected disease. Different staining patterns will help diagnose an autoimmune bullous disease.

For which blistering skin conditions is indirect immunofluorescence tested?

Indirect immunofluorescence is used to diagnose the following autoimmune blistering diseases:

• Pemphigus vulgaris
• Leaf Pemphigus
• Paraneoplastic pemphigus
• Immunoglobulin (Ig) A pemphigus
• Linear bullous IgA skin disease and chronic bullous disease of childhood
• Dermatitis herpetiform
• Bullous pemphigoid
• Gestational pemphigoid
• Mucous membrane pemphigoid
• Acquired epidermolysis bullosa

• Bullous systemic lupus erythematosus.

The laboratory method

Preparation of the patient's serum for the test.

1. 5–10 ml of blood is drawn and collected in a tube.
2. The blood is allowed to coagulate.
3. The blood sample is centrifuged to separate the serum from the clot.
4. The serum is divided into parts and frozen until tested.

The indirect immunofluorescence test

1. The tissue is sliced 4 to 6 microns thick for tissue substratum slides (this can be done in a laboratory, or slides can be obtained commercially).
2. Several slides with different tissue substrates are prepared.
3. Tissue sections are incubated with patient serum for 30 minutes and a doubling dilution is made if titers are required.
4. Slides are washed to remove unbound primary antibodies.
5. The slides are incubated for another 30 minutes with secondary antibodies containing a fluorescent dye.
6. Each slide is mounted under a coverslip.
7. Each slide is examined using fluorescence microscopy.

Interpret the results of indirect immunofluorescence.

Immunofluorescent slides are examined for the presence of autoantibodies through patterns of immunity. statement. Results are subjective and indirect immunofluorescence cannot be used reliably to monitor disease severity and treatment.

The different staining patterns seen with indirect immunofluorescence include:

• Intercellular space (ICS) staining pattern
• basement membrane zone (BMZ) staining pattern.

The sensitivity of a diagnostic tool refers to its ability to accurately identify people with the disease (ie, produce a true positive result). The specificity of a diagnostic tool refers to its ability to accurately identify those who do not have the disease (ie, produce a truly negative result).

Occasionally a test can also produce false results. Therefore:

• The result can be negative even if the patient has the disease (ie, produces a false negative result).
• The result may be positive in a patient without the disease (ie, produce a false positive result).

Immunofluorescent patterns of skin diseases.

The immunofluorescent patterns observed in the following skin diseases are described below.

Pemphigus vulgaris

At pemphigus foliaceous, indirect immunofluorescence shows:

• An ICS staining pattern with IgG or C3 deposits
• A sensitivity of 81–87% [4.7]
• A specificity of 81% [7].

Leaf Pemphigus

At pemphigus foliaceous, indirect immunofluorescence shows:

• ICS IgG or C3 standard
• 60-86% [4,7].

Paraneoplastic pemphigus

In pemphigus, indirect immunofluorescence shows:

• C3 and IgG antibodies
• A sensitivity of the 80%. [3]
• A specificity of 83% [3].

Bullous pemphigoid

In bullous pemphigoid, indirect immunofluorescence shows:

• A linear BMZ staining pattern with IgG and C3 deposits
• A sensitivity of the 70% [3].

Mucous membrane pemphigoid

In mucous membrane pemphigoid, indirect immunofluorescence shows:

• A staining pattern of BMZ with IgG or IgA antibodies
• A sensitivity of the 20%. [3].

Gestational pemphigoid

In pemphigoid gestationis, indirect immunofluorescence shows:

• Deposits C3
• A sensitivity of 93% [3].

Acquired epidermolysis bullosa

In epidermolysis bullosa acquisita, indirect immunofluorescence shows:

• A linear BMZ staining pattern with IgG antibodies
• A sensitivity of the 50%. [3].

Herpetiform dermatitis

In dermatitis herpetiformis, indirect immunofluorescence shows:

• IgA anti-endomysial antibodies
• A sensitivity of the 80%. [3].

Linear IgA bullous dermatosis and chronic bullous disease of childhood

In linear IgA bullous dermatosis and chronic bullous disease of childhood, indirect immunofluorescence shows:

• BMZ with IgA antibodies
• A sensitivity of the 50%. [3].

bullous lupus erythematosus

In bullous systemic lupus erythematosus, indirect immunofluorescence shows a linear pattern of BMZ with IgG antibodies.