Neutrophil priming, caused by cell membranes and microvesicles in packed red blood cell units, is abrogated by leukocyte depletion at collection.
BACKGROUND AND OBJECTS: Lipids with platelet activating factor (PAF)-like activity in supernatant of packed red blood cells (PRBC) cause priming of the neutrophil respiratory burst. This effect increases with length of storage. Washing of PRBC has been considered as a means to eliminate this effect; however, the role of the cellular component was not evaluated independently of the supernatant. The source of the inflammatory lipids of the supernatant is likely to be cell membranes altered during ageing in storage and therefore, washing will not eliminate neutrophil priming caused by transfusion of aged PRBC units. The ability of washed PRBC to prime mononuclear cells for another known effect of PAF, the production of IL-8, and the probability that this lipid activity is present on microparticles in PRBC supernatant were also investigated.


METHODS
At collection 10 units of whole blood were split into two equal aliquots one filtered and one unfiltered. PRBC were prepared and stored at 4 degrees C in CPD-AS5. Each week, fresh neutrophils were incubated with samples of washed PRBC and fixed. Change in CD11b, a marker known to increase on the surface of primed neutrophils, was determined by flow cytometry. To determine whether neutrophil priming ability of PRBC supernatant is contained on microvesicles, centrifuged and uncentrifuged supernatant samples were incubated with fresh neutrophils and change in CD11b expression was determined. Plasma IL-8 levels were also measured after exposure of monocytes from fresh whole blood to filtered and unfiltered washed PRBC with and without the addition of fMLP.


RESULTS
Washed PRBC caused a 50-116% increase in CD11b neutrophil surface expression over baseline expression. Filtration of whole blood at collection reduced this CD11b up-regulation by 25-34%. Reduction of priming ability by filtration began on the day of collection and persisted for the storage life of the units. Centrifugation resulted in a reduction of CD11b up-regulation of 11-28% compared with unspun supernatant. Incubation of unfiltered PRBC resulted in priming of mononuclear leukocytes for IL-8 production with a 73-109% increase over baseline, but no increase over baseline was seen for incubation with filtered blood.


CONCLUSIONS
Washing does not eliminate the ability of PRBC units to prime neutrophils and mononuclear cells, because the cellular component of PRBC, in addition to the supernatant, induces priming. Leukodepletion filters significantly reduce these effects compared with unfiltered PRBC. The in vitro beneficial effect of filtration lasts for the shelf life of 42 day units. The ability of PRBC supernatant to prime neutrophils is present on microvesicles.
Cardo
Lisa J
LJ
Walter Reed Army Institute of Research, Department of Blood Research, Transfusion Medicine Branch, Silver Spring, MD 20910-7500, USA. Lisa.Cardo@us.army.mil
Wilder
Donna
D
Salata
Jeanne
J
D001781:Blood Component Removal / Q000379:methods; D001793:Blood Preservation / Q000379:methods; D039481:CD11b Antigen / Q000096:biosynthesis; D004912:Erythrocytes; D005374:Filtration; D006801:Humans; D007249:Inflammation; D016209:Interleukin-8 / Q000096:biosynthesis / Q000378:metabolism; D007962:Leukocytes / Q000166:cytology* / Q000378:metabolism; D007963:Leukocytes, Mononuclear / Q000166:cytology; D008954:Models, Biological; D009000:Monocytes / Q000378:metabolism; D009504:Neutrophils / Q000166:cytology*; D013048:Specimen Handling / Q000379:methods*; D013696:Temperature
D039481:CD11b Antigen; D016209:Interleukin-8