Reticulocyte - Process

Process

To accurately measure reticulocyte counts, automated counters that use lasers mark cell samples with fluorescent dye that marks RNA and DNA (such as thiazole orange or polymethine). This distinguishes reticulocytes as the middle ground of dye response to laser light, between red blood cells (which have neither RNA nor DNA) and lymphocytes (which have a large amount of DNA, unlike reticulocytes).

The specimen requirement for a reticulocyte count is EDTA anti-coagulated whole blood (lavender-top bottle if using the Vacutainer, Vacuette or Monoject systems; red-top if using the S-Monovette system).

Reticulocytes appear slightly bluer than other red cells when looked at with the normal Romanowsky stain. Reticulocytes are also slightly larger, which can be picked up as a high MCV (mean corpuscular volume) with a full blood count done by a trained medical scientist, who has specialized in hematology, or a machine.

Flowcytometry for mouse reticulocytes: One can use a cell-permeable thiazole orange dye (see above) to stain for reticulocytes' residual RNA in conjunction with DRAQ5 DNA-only dye (reticulocytes have no DNA and are, thus, DRAQ5-negative) and Ter119 (glycophorin-A) that is a marker of erythroid lineage. (Thiazole orange dye binds to nucleic acids of both DNA and RNA).