title: Increased anticoagulant osmolality improves separation of leukocytes from red blood cells (RBC).
abstract: 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 granulocytes are removed less effectively, 500-800 x 10(6) or more remaining in the RBC unit. The aim was to improve the separation efficacy by collecting the blood in an hyperosmolar anticoagulant, followed by BAT separation. It was expected that the red cells would shrink, thereby increasing their density, while the granulocytes would not change volume and density.
  
  
  METHODS
  18 donors were included in the study, 12 in the test group and 6 in the control group. CPD-SAGM bags were used, with a modification of the anticoagulant by removal of 20-ml CPD from all units and addition of 20-ml mannitol (test group) or 20 ml of isotonic saline (control group). 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 gated 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 the 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 concentration was slightly 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-day storage. It was possible to prepare platelet concentrates with good yield using the pooled-BC method.
  
  
  CONCLUSIONS
  This study indicates that considerable improvement in the BAT procedure can be obtained if the anticoagulant is made hypertonic by the addition of mannitol. This is achieved without altering the already low levels of MNC and keeping the same quality.
authors.affiliation: Department of Clinical Immunology and Transfusion Medicine, University Hospital, Uppsala, Sweden. folke.knutson@klinimm.uu.se
authors.forename: F
authors.initials: F
authors.lastname: Knutson
authors.forename: H
authors.initials: H
authors.lastname: Lööf
authors.forename: C F
authors.initials: CF
authors.lastname: Högman
mesh_terms: D000925:Anticoagulants / Q000737:chemistry*; D001781:Blood Component Removal / Q000379:methods* / Q000592:standards; D002469:Cell Separation / Q000379:methods* / Q000592:standards; D004912:Erythrocytes; D006098:Granulocytes; D006801:Humans; D007962:Leukocytes; D008353:Mannitol / Q000494:pharmacology; D009994:Osmolar Concentration; D012965:Sodium Chloride / Q000494:pharmacology
chemical_list: D000925:Anticoagulants; D008353:Mannitol; D012965:Sodium Chloride