Influence of cell‐free DNA in plasma on real‐time polymerase chain reaction for determination of residual leucocytes in platelet concentrates

Abstract

Real-time quantitative (RQ) polymerase chain reaction (PCR) can be used to determine the number of residual leucocytes in leucocyte-reduced platelet concentrates (LR-PCs), which should contain < 3.3 leucocytes/ micro l. In this study we investigated the extent to which cell-free DNA, known to be present in plasma, might interfere with this determination. In this study, RQ-PCR was employed to determine the following: the influence of filtration of platelet concentrates (PCs) on the amount of cell-free DNA; the variation in concentration of cell-free DNA between the buffy coats (BCs) of different donors; and the amount of cell-free DNA during storage and processing of whole blood. PCs were sampled before and after filtration (n = 5), BCs were sampled (n = 100) and whole blood units were sampled < 2 h and 16-20 h after collection, and the BCs were also sampled after processing the whole blood (n = 10). Samples were centrifuged to obtain cell-free plasma in which the amount of cell-free DNA was determined using an RQ-PCR for the albumin gene. The amount of cell-free DNA was not influenced by filtration of the PCs [1.7 +/- 0.8 vs. 1.5 +/- 0.8 leucocyte-equivalents (eq)/ micro l]. However, the amount of cell-free DNA in plasma of the BCs varied considerably, from 0.1 to 18.2 leucocyte-eq/ micro l (median = 1.5 leucocyte-eq/ micro l; mean +/- SD: 2.2 +/- 2.4 leucocyte-eq/ micro l). In 18% of the BCs the amount cell-free DNA was > 3.3 leucocyte-eq/ micro l. The amount of cell-free DNA increased during storage, from 0.3 +/- 0.3 leucocyte-eq/ micro l (< 2 h after collection) to 0.9 +/- 0.6 leucocyte-eq/ micro l (16-20 h after collection) and, after processing the whole blood, to 2.0 +/- 2.0 leucocyte-eq/ micro l. Variable amounts of cell-free DNA in plasma will interfere if RQ-PCR is applied to estimate leucocyte numbers in leucocyte-reduced PCs.