It is widely accepted that different blood collection conditions, including anticoagulants, influence leukocyte phenotype and function. Buffy Coats originated from a donated whole blood bag unit are commonly used in immunological research as a source of leukocytes. They are a residual product of healthy donor whole blood processing. The preservative solution present in the blood bag unit and consequently in the derived Buffy Coat is Citrate-Phosphate-Dextrose (CPD), in which citrate is the anticoagulant. There is a lack of information on the possible difference in the functionality of leukocytes from Buffy Coats originated from a blood bag unit vs leukocytes isolated from blood collection tubes with various anticoagulants.

Herein, we aimed at studying monocyte function when the monocytes are isolated from Buffy Coats originated from a blood bag unit vs blood collection tube containing EDTA, CPD with adenine (CPDA), or sodium citrate. The function of monocytes, isolated 20 h after blood collection, to present lipid antigens to invariant Natural Killer T (iNKT) cells was investigated. iNKT cells are activated by lipids bound to CD1d, a non-polymorphic MHC-class I-like molecule, present on the surface of antigen-presenting cells.

A striking result showed that monocytes isolated from EDTA blood tubes have a lower capacity to present lipid antigens to iNKT cells than monocytes isolated from Buffy Coats originated from a blood bag unit. No differences were found between monocytes isolated from sodium citrate or CPDA and the ones isolated from Buffy Coats originated from a blood bag unit. This was accompanied by a decrease in viability of the EDTA-isolated monocytes. Expression of the surface markers CD1d and CD86 was higher for monocytes isolated from EDTA than those isolated from Buffy Coats.

In conclusion, EDTA-containing blood tubes are not the ideal choice of anticoagulant for monocyte antigen presentation assays. We advise that the blood collection condition and the time between biospecimen collection and analysis should be carefully considered when designing experimental procedures.