The blood used to isolate cells for analysis in HLA labs often cannot be processed immediately and may be drawn in a variety of anti-coagulants. We evaluated the effect of the time between draw and cell enrichment, and the effects of drawing blood in different anti-coagulants, using RosetteSep™ cell enrichment kits and the SepMate™-50 tube.Blood was collected in heparin, ACD, or EDTA and enriched for lymphocyte subsets (T cells, B cells, Total Lymphocytes (TL), and Lymphoid (CD3+) cells) or myeloid cells using RosetteSep™ cell enrichment cocktails either immediately (0hr) or after storing the blood at room temperature for 48hr. Samples were incubated with the cocktail for 10min, layered over a buoyant density medium in a SepMate™ tube, and spun for 10min with the brake off. Purity and recovery of cells was determined by flow cytometry.The purity and recovery of lymphocyte subsets and myeloid cells enriched from blood drawn in heparin or ACD were not significantly different (paired t tests, p>0.05), with the exception of TL enrichment after 48hr, when the purity of TL enriched from blood drawn in heparin was slightly higher. The purity and recovery of cell subsets enriched from blood drawn in EDTA were frequently lower than the same cell subsets enriched from the same samples collected in heparin or ACD, particularly when enriching B cells.Lymphocyte subsets and myeloid cells can be enriched using RosetteSep™ and SepMate™ up to 48hr post blood-draw, with minimal effects on purity and recovery. The highest purities and recoveries are most consistently obtained using blood collected in heparin or ACD. The use of EDTA as an anti-coagulant is not recommended, particularly if the sample will not be processed soon after draw or if the desired cell subset includes B cells. Approaches to mitigate the effects of collecting blood in different anti-coagulants, such as altering storage temperatures, will be further explored.Peters: STEMCELL Technologies Inc: Employee. Fadum: STEMCELL Technologies Inc: Employee. Zhang: STEMCELL Technologies Inc: Employee. McQueen: STEMCELL Technologies Inc: Employee. Thomas: STEMCELL Technologies Inc: Employee.