Evaluation of a New Single-Gradient, Ficoll-Hypaque with Hypotonic Lysis Method for Leukocyte Separation

Abstract

Objective: Separation techniques for radiolabeled leukocytes possess inherent problems: procedure length, leukocyte numbers and contaminates (e.g., platelets and erythrocytes). A new leukocyte separation technique is compared to current methods.

Methods: These four leukocyte separation methods were compared: volex sedimentation (VS), volex sedimentation with hypotonic lysis (VL), Ficoll-Hypaque two density gradients (FH) and a new single density gradient Ficoll-Hypaque with hypotonic lysis (FL). CBC data was used to determine the cellular composition of each method. Time to perform each method was also documented. In vitro viability of the leukocytes separated by the FL method was compared to the FH separation method using Chemotaxis and trypan blue staining.

Results: The VS and VL methods demonstrated the largest number of leukocytes and platelets. The FH and FL techniques yielded reduced numbers of platelets and erythrocytes with a high percentage of neutrophils present (average ~94%). FH required the most time (3 hr) for the separation completion, whereas VL required the least time (90 min). FH and FL methods yielded viable leukocyte populations by Chemotaxis and trypan blue staining evaluation.

Conclusions: The new FL separation method compares favorably to other leukocyte separation techniques. The reduction in the number of cell contaminants, increased percentage of neutrophils present, a viable leukocyte population, and the time required to perform the method are all factors which make the FL technique attractive.