Immunofluorescence

Definition
Immunofluorescence is a laboratory technique to identify specific antibodies or antigens. Antibody identification is usually performed on blood (serum).

How the test is performed

Blood is drawn from a vein, usually on the inside of the elbow or the back of the hand. The puncture site is cleaned with antiseptic, and an elastic band is placed around the upper arm to apply pressure and restrict blood flow through the vein. This causes veins below the band to fill with blood.

A needle is inserted into the vein, and the blood is collected in an air-tight vial or a syringe. During the procedure, the band is removed to restore circulation. Once the blood has been collected, the needle is removed, and the puncture site is covered to stop any bleeding.

For an infant or young child, the area is cleansed with antiseptic and punctured with a sharp needle or a lancet. The blood may be collected in a pipette (small glass tube), on a slide, onto a test strip, or into a small container. Cotton or a bandage may be applied to the puncture site if there is any continued bleeding.

Immunofluorescence is a laboratory technique. The exact technique may vary depending on the specific antibody that is being investigated and between different laboratories.

In general, cells, tissue, or some other substance is placed onto microscope slides. A small amount of sample containing antibodies (typically serum, a liquid portion of blood) is placed over the cells or tissue, allowing the antibodies that are specific for the particular tissue or cellular antigens to bind.

The serum is washed away, and a second antibody that binds to human antibodies (often made in another animal species such as rabbits or goats) is applied to the slide. This second antibody has a fluorescent dye chemically linked to it. If the person’s serum has antibodies that bind to the tissue or cells, a bright fluorescence can be seen by use of a special microscope.

How to prepare for the test
No special preparation is necessary.

For infants and children:
The preparation you can provide for this test depends on your child’s age and previous experiences. For specific information regarding how you can prepare your child, see the following topics:

     
  • infant test or procedure preparation (birth to 1 year)  
  • toddler test or procedure preparation (1 to 3 years)  
  • preschooler test or procedure preparation (3 to 6 years)  
  • schoolage test or procedure preparation (6 to 12 years)  
  • adolescent test or procedure preparation (12 to 18 years)

How the test will feel
When the needle is inserted to draw blood, some people feel moderate pain, while others feel only a prick or stinging sensation. Afterward, there may be some throbbing.

Why the test is performed
This test is used to detect and roughly measure the amount of antibodies in the blood.

Normal Values
A negative test result is normal.

What abnormal results mean

Immunofluorescence is often used to detect and quantitate antinuclear antibody (ANA). These are antibodies that the body makes against itself in some autoimmune diseases like SLE. The presence of these antibodies aid in the diagnosis and management of these diseases.

The test may also be used to detect and measure the quantity of antibodies against microorganisms such as Coxiella Brunetti, Rickettsii or Colorado tick virus. A positive result in this case indicates that the body’s immune system has seen the pathogen at some point in the past. Variations on this test can be used to better define the timing of the past exposure.

This test may also be used to identify various microorganisms in fresh samples or after culturing them in the laboratory. In this case, antibodies that are known to be specific for particular pathogens are used on infected cells. This test is also known as a direct fluorescent antibody (DFA) test.

Additional diseases for which the test may be performed:

     
  • Colorado tick fever  
  • focal segmental glomerulosclerosis  
  • infectious mononucleosis (CMV)  
  • infectious mononucleosis (EB)  
  • Lyme disease  
  • Lyme disease; primary  
  • Lyme disease, secondary  
  • Lyme disease, tertiary  
  • minimal change disease  
  • pemphigus vulgaris  
  • Q fever (early)  
  • Q fever (late)  
  • rocky mountain spotted fever

What the risks are

     
  • excessive bleeding  
  • fainting or feeling light-headed  
  • hematoma (blood accumulating under the skin)  
  • infection (a slight risk any time the skin is broken)  
  • multiple punctures to locate veins

Special considerations
Veins and arteries vary in size from one patient to another and from one side of the body to the other. Obtaining a blood sample from some people may be more difficult than from others.

Johns Hopkins patient information

Last revised: December 6, 2012
by Dave R. Roger, M.D.

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