Development of white blood cell fragments, during the preparation and storage of platelet concentrates, as measured by using real-time polymerase chain reaction.
OBJECTIVE
White blood cell (WBC) fragments may cause human leucocyte antigen (HLA) immunization in recipients. We investigated the occurrence and production of WBC fragments in platelet concentrates (PCs) and plasma units, during storage and filtration, by using real-time polymerase chain reaction (PCR) and flow cytometry.


METHODS
To study the occurrence of WBC fragments, 'male' WBCs were spiked into double-filtered 'female' PCs in a concentration series of 0.03-100 WBCs/microl (n = 4 per level). To study the production of WBC fragments, 'male' WBCs were spiked into 'female' plasma units to 4 x 10(9) WBCs/l and stored at room temperature prior to filtration (n = 4 per storage time; t = 0, 24 or 48 h). DNA was measured by both albumin real-time PCR and Y real-time PCR. Intact WBCs were counted by using flow cytometry. The number of WBC fragments was calculated by subtracting cell-free DNA (real-time PCR on supernatant) and intact WBCs (flow cytometry) from the total DNA amount (real-time PCR).


RESULTS
Spiking of 'male' WBCs into 'female' PCs showed that the Y real-time PCR is linear and has a reproducible quantitative range down to 0.03 WBC/microl, but that the albumin-PCR, in unspiked samples, revealed a total of 6-10 WBC equivalents/microl (eq/microl). After centrifugation, half of this was observed as cell-free DNA in the supernatant, suggesting that the remaining DNA is derived from WBC fragments. The number of intact WBCs, amount of cell-free DNA and number of WBC fragments after filtration increased significantly when filtration was delayed for up to 48 h, from 0.1 WBC/microl, 1.3 WBC eq/microl and 0.6 WBC eq/microl at t = 0 h to 25 WBC/microl, 38 WBC eq/microl and 57 WBC eq/microl at t = 48 h, respectively.


CONCLUSIONS
WBC fragments occur in WBC-reduced PCs and increase when products are stored, prior to filtration, up to levels that are equivalent to the amounts of intact WBCs that induce HLA immunization (i.e. > 5 x 10(6)/unit).
Dijkstra-Tiekstra
M J
MJ
Sanquin Blood Bank North-west Region, Amsterdam, the Netherlands. m.dijkstra@sanquin.nl
van der Schoot
C E
CE
Pietersz
R N I
RN
Huijgens
P C
PC
van der Meer
P F
PF
Reesink
H W
HW
D001793:Blood Preservation; D004247:DNA / Q000097:blood; D005260:Female; D005374:Filtration; D006680:HLA Antigens / Q000276:immunology; D006801:Humans; D007958:Leukocyte Count; D047589:Leukocyte Reduction Procedures / Q000592:standards*; D007962:Leukocytes / Q000473:pathology; D008297:Male; D017713:Platelet Transfusion / Q000009:adverse effects / Q000592:standards*; D016133:Polymerase Chain Reaction
D006680:HLA Antigens; D004247:DNA