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False-Positive IMMY® Serum Cryptococcal Antigen Lateral Flow Assay Results Due to Improper Use of Titration Buffer
Asmus Tukundane1,2, Wilber Bakka1, Mercy Amanige1
1Infectious Diseases Institute, College of Health Sciences, Makerere University, Kampala P.O. Box 22418, Uganda.
Insights
Incorrect use of titration diluent in cryptococcal antigen (CrAg) lateral flow assays (LFAs) can cause false positives. Adhering to manufacturer protocols is crucial for accurate Cryptococcus diagnosis and patient management.
Area of Science:
- Clinical diagnostics
- Medical laboratory science
- Infectious disease diagnostics
Background:
- The IMMY cryptococcal antigen (CrAg) lateral flow assay (LFA) is vital for Cryptococcus detection.
- False-positive results can occur due to procedural or reagent errors, impacting diagnostic accuracy.
- Careful assay operation is essential to prevent misdiagnosis and unnecessary treatment.
Purpose of the Study:
- To investigate the cause of false-positive CrAg LFA results in two referred patients.
- To identify the specific procedural error leading to diagnostic misinterpretation.
- To emphasize the importance of adhering to manufacturer's instructions for CrAg LFA.
Main Methods:
- Repeat CrAg LFA testing using specimen diluent according to manufacturer's protocol.
- Semi-quantitative CrAg LFA testing and control assays.
- Serial dilutions to assess the impact of incorrect diluent use.
Main Results:
- Repeat tests with correct specimen diluent yielded negative results.
- Inconsistent use of titration diluent instead of specimen diluent reproducibly generated positive results.
- False positivity persisted up to a 1:80 dilution when titration diluent was misused.
Conclusions:
- Inappropriate use of titration diluent instead of specimen diluent is a likely cause of false-positive CrAg LFA results.
- This error may stem from the absence of a blocking agent in titration diluent, which normally neutralizes heterophilic antibodies.
- Strict adherence to procedural guidelines is critical for accurate diagnostic testing and effective patient management.
Abstract:
The IMMY cryptococcal antigen (CrAg) lateral flow assay (LFA) is a reliable diagnostic tool for Cryptococcus detection, but false-positive results may arise from procedural or reagent-related errors, underscoring careful operation of the assay to ensure diagnostic accuracy and prevent unnecessary treatment. Two patients who were initially reported as CrAg-positive by a peripheral laboratory were referred to Kiruddu Hospital in Kampala, Uganda, for clinical assessment and confirmatory testing. Repeat tests were conducted using specimen diluent following the manufacturer's protocol, resulting in negative results. Semi-quantitative CrAg LFA testing and a series of control assays were performed to identify the source of error. We were able to consistently reproduce positive results when the titration diluent was inappropriately used instead of the specimen diluent. Serial dilutions confirmed persistent false positivity up to 1:80 when inappropriately using the titration diluent, while all dilutions that appropriately started with the specimen diluent remained negative. We hypothesize that the incorrect use of titration diluent instead of specimen diluent led to false-positive CrAg LFA results due to the absence of the blocking agent that neutralizes heterophilic antibodies. Procedural errors can lead to diagnostic misinterpretation and serious consequences in clinical management, emphasizing the importance of adherence to manufacturer's instructions.
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