Increased anticoagulant osmolality improves separation of leukocytes from red blood cells (RBC)

Background: The bottom-and-top (BAT) procedure separates the buffy coat (BC ) from plasma and red blood cells (RBC). The contents of mononuclear cells (MNC) remaining in the RBC are about 1 x 10(6) cells/unit, whereas the gran ulocytes are removed less effectively, 500-800 x 106 or more remaining in t he RBC unit. The aim was to improve the separation efficacy by collecting t he blood in an hyperosmolar anticoagulant, followed by BAT separation. It w as expected that the red cells would shrink, thereby increasing their densi ty, while the granulocytes would not change volume and density. Study design and methods: 18 donors were included in the study, 12 in the t est group and 6 in the control group. CPD-SAGM bags were used, with a modif ication of the anticoagulant by removal of 20-ml CPD from all units and add ition of 20-ml mannitol (test group) or 20 mi of isotonic saline (control g roup). The collected blood units were cooled on butanediol plates for 2-4 h , then centrifuged and separated into components. The levels of leukocytes in the whole blood, the BC and the RBC were determined by flow cytometry ga ted for intact CD45 + cells. A number of other tests were performed during 42-day storage. Results: The plasma yield was slightly higher in the test group than in the control group (ns). The contents of leukocytes (CD 45 + intact cells) in t he RBC units were 32 +/- 20 x 10(6) in the test group and 573 +/- 241 x 10( 6) in the control group. The numbers of MNC were 1.2 +/- 0.6 x 10(6) and 2. 6 +/- 1.8 x 10(6), respectively. The RBC 2,3-DPG con centration was slightl y better maintained in the test group at day 7 of refrigerated storage (p = 0.0027), but most other tested parameters showed no difference during 42-d ay storage. It was possible to prepare platelet concentrates with good yiel d using the pooled-BC method. Conclusion: This study indicates that considerable improvement in the BAT p rocedure can be obtained if the anticoagulant is made hypertonic by the add ition of mannitol. This is achieved without altering the already low levels of MNC and keeping the same quality. (C) 2000 Elsevier Science Ltd. All ri ghts reserved.