What is “the RFFIT”?
The acronym RFFIT stands for Rapid Fluorescent Focus Inhibition Test. The test measures the ability of antibodies that may be present in a sample to neutralize and block rabies virus from infecting the cells used in the test. These antibodies are called rabies virus neutralizing antibodies (RVNA). In the test, serum (the non-cellular portion of a blood sample) is first diluted fivefold (1 part serum in 4 parts diluent). Further (serial fivefold) dilutions are performed, each of which contain less and less of the sample. The serum dilutions are mixed with a standard amount of live rabies virus and incubated. If RVNA are present in the sample, they will bind to the virus. Tissue culture cells are then added and incubated with the test sample and virus. Whatever rabies virus that may not have been neutralized by the antibody in the sample will then infect the cells. These foci of infection in the cells can then be seen under the fluorescent microscope. If there are a lot of infected cells, there is very little antibody; conversely, if antibodies are high, the cells will have very little evidence of infection. The endpoint titer is calculated from the percent of virus-infected areas observed within the wells containing the various dilutions of the sample on the slide.
What does your result tell you?
The RFFIT result can be reported in either a titer which is a ratio (e.g., 1:50) or as a standardized concentration represented as international units (IU) per mL of serum (e.g., 0.5 IU/mL). The IU value is calculated from the titer by comparing it against a standard reference serum. We use the following formula: sample titer divided by the reference serum titer, multiplied by the IU/mL value of the reference serum.
Because the RFFIT test is a biological system using live cells, infectious virus, and antibodies, the reference serum can vary in titer level for each batch of testing (within an established acceptable range). Therefore the calculation of IU/mL depends on the titer of the reference serum measured in the batch tested. In general, you can take the titer value divided by 100 to get a rough estimate of the IU/mL value. To obtain the exact value you must use the calculation with the reference serum titer value that resulted from that batch of tests.
According to World Health Organization guidelines, a rabies antibody level of greater than or equal to 0.5 IU/mL demonstrates an adequate response to vaccination (1). If the level falls below this value, a booster dose of rabies vaccine may be recommended for people who are at frequent risk of rabies virus exposure. In contrast, the ACIP guidelines state that evidence of complete neutralization at a serum dilution of 1:5 in RFFIT testing (corresponding to 0.1-0.2 IU/mL in our laboratory) is considered an adequate response to rabies vaccination (2). The lowest antibody level that can be accurately and precisely measured by the RFFIT in our laboratory is 0.1 IU/mL; below this level, there is uncertainty as to the specificity of the result. Because the ACIP level is close to the assay threshold, the level of 0.5 IU/mL is more conservative for guiding human vaccination decisions and applicable in most situations.
Points that should be considered as to whether a person should receive a booster dose of rabies vaccine when their antibody level falls below 0.5 IU/mL are: