In clinical organ transplantation, flow cytometry crossmatching can be performed on total blood with a hemolysis step or after a preliminary mononuclear cell separation step using a standard Ficoll-Hypaque protocol. Here, we compared the Ficoll-Hypaque step with a faster technique for isolating mononuclear cells (the SepMate tube), using the same samples (collected and stored at room temperature for 0, 24, 48 or 72 hours). We found that the SepMate separation protocol is easily applied to flow cytometry crossmatching (with or without pronase treatment), provided that the samples have been stored at room temperature for 48 hours or less. Conversely, the Ficoll-Hypaque protocol should be used if the samples have been stored for more than 48 hours at room temperature.
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