How is a Monospot (Mononucleosis spot test) performed?

How to Perform a Monospot Test

The Monospot test is a rapid heterophile antibody test performed on a serum or plasma sample using red cell or latex agglutination to detect antired cell antibodies produced during EBV infection. 1

Specimen Collection and Handling

• Collect a serum specimen as soon as possible after symptom onset for optimal diagnostic accuracy. 2
• The test can be performed on either serum or plasma samples obtained through standard venipuncture. 1
• If the initial Monospot test is negative but clinical suspicion remains high, the test can be repeated on a serum specimen obtained 7-10 days later, at which time the heterophile antibodies are more likely to be positive if EBV infection is present. 3

Test Methodology

• The Monospot test uses red cell or latex agglutination assays that detect heterophile antibodies (antired cell antibodies) produced as part of the polyclonal antibody response during EBV infection. 1
• The test is rapid, inexpensive, and can be performed from the onset of symptoms of infectious mononucleosis. 1
• Results are typically available within minutes, making it an efficient point-of-care diagnostic tool. 4

Test Performance Characteristics

• In adolescents and young adults (15-24 years), the Monospot test has 87% sensitivity and 91% specificity for diagnosing acute EBV infection. 4
• The test has high specificity and sensitivity in adolescents but performs poorly in children under 4 years of age, where it has low sensitivity and low negative predictive value. 1
• False-negative results occur in approximately 10% of cases, most commonly in younger children and during the first week of illness in adults. 3, 5

Critical Interpretation Points

• A positive Monospot test is considered diagnostic for EBV infection and no further EBV-specific testing is required. 3, 5
• A negative Monospot test does not rule out EBV infection, particularly in children under 10 years or during the first week of symptoms. 3, 5
• When the Monospot is negative but clinical suspicion remains high (fever, pharyngitis, posterior cervical lymphadenopathy, atypical lymphocytosis >10%), perform EBV-specific serologic testing for VCA IgM, VCA IgG, and EBNA antibodies on the same sample. 3, 5, 2

Common Pitfalls to Avoid

• Do not rely solely on a negative Monospot test to exclude infectious mononucleosis, especially in children, as this leads to misdiagnosis. 5
• The test should not be used as the primary diagnostic tool in children under 4 years of age; proceed directly to EBV-specific serology in this population. 1
• False-positive results may occur in patients with leukemia, pancreatic carcinoma, viral hepatitis, CMV infection, other viral infections, autoimmune disease, and hematological malignancies. 5, 1
• Heterophile antibodies may take time to develop, so timing matters—testing too early in the illness course increases the risk of false-negative results. 5, 4

Supporting Laboratory Findings

• Obtain a complete blood count with differential to assess for >40% lymphocytes and >10% atypical lymphocytes, which supports the diagnosis when present. 4
• The presence of elevated liver enzymes increases clinical suspicion for infectious mononucleosis even when the heterophile antibody test is negative. 4