CD40LG and infection: The HI assay has some limitations that make it is impractical, such as: (a) each serotype requires a different optimal pH of RBCs and therefore requires the use of multiple pH buffers; (b) it is unable to discriminate infections either between DENV and other related Flaviviruses or between immunoglobulin isotypes (IgM vs. IgG); and (c) it may require chemical and heating pre-treatment to remove nonspecific inhibitors in the clinical specimen [119,215].