Dijkstra-Tiekstra M J, Pietersz R N I, Reesink H W, van der Schoot C E
Sanquin Blood Bank north-west Region, Amsterdam, the Netherlands.
Vox Sang. 2004 Feb;86(2):130-5. doi: 10.1111/j.0042-9007.2004.00402.x.
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.
实时定量（RQ）聚合酶链反应（PCR）可用于测定白细胞滤除血小板浓缩物（LR-PCs）中残留白细胞的数量，LR-PCs中白细胞含量应<3.3个/微升。在本研究中，我们调查了血浆中存在的游离DNA可能干扰该测定的程度。本研究采用RQ-PCR测定以下内容：血小板浓缩物（PCs）过滤对游离DNA量的影响；不同供者的 Buffy 层（BCs）中游离DNA浓度的差异；以及全血储存和处理过程中游离DNA的量。
在过滤前后采集PCs样本（n = 5），采集BCs样本（n = 100），并在全血采集后<2小时和16 - 20小时采集全血样本，在处理全血后也采集BCs样本（n = 10）。将样本离心以获得无细胞血浆，使用针对白蛋白基因的RQ-PCR测定其中游离DNA的量。
PCs过滤对游离DNA量无影响[1.7±0.8 vs. 1.5±0.8白细胞当量（eq）/微升]。然而，BCs血浆中游离DNA的量差异很大，范围为0.1至18.2白细胞-eq/微升（中位数 = 1.5白细胞-eq/微升；平均值±标准差：2.2±2.4白细胞-eq/微升）。在18%的BCs中，游离DNA量>3.3白细胞-eq/微升。游离DNA量在储存过程中增加，从采集后<2小时的0.3±0.3白细胞-eq/微升增加到采集后16 - 20小时的0.9±0.6白细胞-eq/微升，在处理全血后增加到2.0±2.0白细胞-eq/微升。
如果应用RQ-PCR估计白细胞滤除PCs中的白细胞数量，血浆中游离DNA量的变化会产生干扰。
