# False-Positive IMMY® Serum Cryptococcal Antigen Lateral Flow Assay Results Due to Improper Use of Titration Buffer

## TL;DR

False-positive results in a Cryptococcus antigen test occurred due to improper use of dilution buffer, highlighting the need for strict adherence to protocols.

## Contribution

The study identifies a specific procedural error involving diluent misuse that leads to false-positive IMMY® CrAg LFA results.

## Key findings

- Using titration diluent instead of specimen diluent caused consistently false-positive results up to a 1:80 dilution.
- False positives were eliminated when specimen diluent was used correctly, as per manufacturer instructions.
- The absence of a blocking agent in the incorrect diluent is hypothesized to cause interference from heterophilic antibodies.

## 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.