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Sputum Direct Fluorescent Antibody (DFA)

What Is a Sputum DFA Test?

Sputum direct fluorescent antibody (DFA) is a laboratory test your doctor may order if you have a lung infection or pneumonia. This test is performed on a sample of your sputum (phlegm) to find specific types of microorganisms (bacteria). It is also known as the direct immunofluorescence test and Legionella DFA.

The sputum DFA test is used to identify two types of bacterium:

  • Legionella,which causes Legionnaires’ disease
  • Mycoplasma pneumonia (M. pneumonia), which causes the less common and less severe disease called "walking pneumonia"

This test is one of the best ways to find out if your respiratory illness is caused by a bacterial infection, and if so, what kind of infection it is. Confirming the diagnosis allows your doctor to provide the right antibiotic treatment.

Why the Test Is Ordered

Your doctor may order the sputum DFA test when he or she sees symptoms of either Legionnaires’ disease (L) or mycoplasma pneumonia (MP), including:

  • chest pain and difficulty breathing (L and MP)
  • dry cough (MP) or coughing up blood (L)
  • mild or high fever (MP)
  • high fever of 102 to 105 F (L)
  • sweating and clammy skin (MP)
  • abdominal pain, nausea, vomiting, and diarrhea (L)
  • headache, muscle aches, and stiffness in the joints (L and MP)
  • loss of appetite, low energy, fatigue (L and MP)
  • ear and eye pain (MP)
  • neck lump, rash, and sore throat (MP)

How to Collect a Sample of Your Sputum

If you are very sick, you may already be in the hospital. If so, the bedside nurse will help you cough up sputum to send to the laboratory for the test. If you have trouble coughing up sputum on your own, the nurse may have you breathe steam.

If you are sick at home, you will need to collect the sputum sample yourself.

Keep in mind, sputum from deep inside your lungs isn’t the same as saliva. Sputum is mucus. It is usually colored and thick in consistency, especially when there is infection in the lungs. Saliva comes from your mouth and is clear-colored and thin.

Plan to collect sputum the first thing in the morning. This makes the test more accurate. Do not eat or drink anything in the morning before collecting your sample. Your doctor will provide a sterile sample cup. Wait until you are ready to collect your sample before opening the lid.

To collect a sputum sample:

  • Brush your teeth and rinse your mouth (don’t use antiseptic mouthwash).
  • Take a couple of very deep breaths.
  • Breathe in deeply again and cough hard until sputum comes up.
  • Spit out the sputum into the sample cup.
  • Keep coughing up sputum until the cup is filled to the marker (approximately one teaspoon).
  • Screw on the cup lid, and wash and dry the outside of the cup.
  • Write your name and the date on the cup label.

Take the sample to the clinic or laboratory, as instructed. The sample can be refrigerated for up to 24 hours, if needed. Do not freeze it or store it at room temperature.

If you can’t cough up sputum, try breathing the steam from boiling water, or take a hot, steamy shower. The sputum must come from deep inside your lungs for the test to be accurate.

If you still cannot cough up sputum, your doctor will do a bronchoscopy to collect sputum directly from the lungs using a bronchoscope.

How to Prepare for Self-Collection

The night before the test, drink lots of fluids, such as water or tea. This will help your body make more sputum overnight. Collecting your sputum in the morning makes the test more accurate. More bacteria are present first thing in the morning.

How the Doctor Collects Sputum

Bronchoscopy is a simple procedure that takes about 30 to 60 minutes. It is often done in the physician’s office, and the patient usually remains awake.

Do not to take medicines that thin your blood the day before your bronchoscopy. These medicines include aspirin, Motrin, Advil, and Aleve. You may take Tylenol if needed for pain. You will also be asked not to eat or drink anything the night before the procedure.

Brochoscopy is performed as follows:

  • A local anesthetic will be sprayed into your nose and throat to numb them.
  • You might be given a sedative to help you relax, or medication to put you to sleep.
  • General anesthesia is not usually needed for bronchoscopy.
  • The bronchoscope is a soft, small-circumference tube with a light and magnifying glass on the end. The doctor feeds the scope through your nose or mouth, and into your lungs.
  • Using the scope, the doctor can see into the lungs and use it to remove sputum.
  • A nurse will attend you during and after the procedure until you are fully awake.
  • For safety, you should have someone else drive you home.


There are no risks in collecting a sputum sample yourself. You might feel light-headed when coughing deeply. Rare risks of bronchoscopy include allergic reactions to sedatives, infection, bleeding, pneumothorax (tearing in the lungs, which results in a little air being released between the lung and chest wall, and usually fixes itself), bronchial spasms (a sudden clenching of the muscles in the bronchioles), and irregular heart rhythms.

How the Test Works

Sputum DFA is a complex test that requires the careful selection of specific antigens and antibodies which are used to indirectly identify the target microorganisms.

An antigen is a protein marker on a bacterium cell. The body’s immune system recognizes antigens as a threat. This triggers production of antibodies (white blood cells) to fight the infection.

In the laboratory, the specific antibodies that correspond to the bacteria’s antigens are chemically linked to fluorescent dye and then added to the sputum sample. The "fluorescent tagged" antibodies bind to the antigens and become visible under the microscope.

There are many causes of pneumonia, as well as other bacterial species of Legionella. It is important that the laboratory selects the exact antigens and corresponding antibodies to make a positive identification.

Test Results

Abnormal results mean that Legionella or M. pneumonia bacteria are present in the sputum. This test is frequently negative during the first days of illness because there are fewer organisms in the sputum. More than one test may be needed to identify the exact cause of symptoms.

About Legionnaires’ Disease

According to the Centers for Disease Control and Prevention (CDC), bacterial infections are among the top three causes of pneumonia. Legionella is a fairly common cause of pneumonia. It is estimated that there are more than 25,000 cases and 4,000 deaths caused by Legionnaires’ disease in the United States each year (CDC, 2011).

Legionella bacteria grow in warm and stagnant water, which can sometimes be found in such places as hot water tanks, swimming pools, hot tubs, or moist duct systems in large buildings. Another common place where this bacterium grows is the water spray systems in grocery store produce departments.

People get infected by breathing Legionella-contaminated mist or moist air. Those over 50 years old are most susceptible to getting Legionnaires’ disease.

Long-Term Outlook

With the right diagnosis and antibiotic treatment, patients usually get better quickly. However, the disease can be severe, leading to problems that include kidney failure, diabetes, chronic obstructive pulmonary disease (COPD), weakened immune system, and death.

Content licensed from:

Written by: Sandy Calhoun Rice
Medically reviewed : George Krucik, MD

This feature is for informational purposes only and should not be used to replace the care and information received from your health care provider. Please consult a health care professional with any health concerns you may have.
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