Plasma‐thrombin cell blocks: Potential source of DNA contamination

Abstract

Background Cell blocks are being used more frequently in cytology for ancillary testing, including molecular diagnostics. There are several different methods of processing cell blocks, with plasma‐thrombin being one of the most common. Plasma is a blood‐derived product and may be a source of DNA. The aim of this study was to determine whether the plasma used for the plasma‐thrombin cell block method has amplifiable DNA that may potentially interfere with molecular testing results. Methods Expired bags of fresh frozen plasma were collected from a blood bank. From each sample, DNA was extracted from a 1‐mL aliquot with the QIAsymphony MIDI kit (Qiagen). The concentration of DNA was measured on a NanoDrop instrument. The amplifiable DNA quality was assessed by polymerase chain reaction (PCR) with primers to generate amplicons of various sizes. Characterization was performed with the AmpFLSTR Identifiler Plus PCR kit with capillary electrophoresis. Results Twenty samples from 20 bags were collected. All samples showed amplifiable DNA despite low DNA concentrations in a few cases. PCR amplification revealed the presence of high‐quality amplifiable DNA (up to 600 base pairs). DNA was amplified at the 16 loci interrogated in all samples tested with the AmpFLSTR Identifiler Plus PCR kit. Conclusions The presence of genomic DNA in plasma may theoretically interfere with results of molecular testing. Particularly in clinical samples with low cellularity, the DNA in plasma may potentially either mask the presence of minute amounts of tumor‐derived DNA or lead to a false‐positive result.

A novel method for making cell blocks with higher cellular yield

“…However, the fibrin could be seen in the PT method just as other studies have reported 20 . One study demonstrated the presence of significant amounts of amplifiable DNA in expired plasma, which can potentially contaminate CB samples prepared with PT 14 . Theoretically, cell‐free DNA and circulating tumor DNA may interfere with molecular tests.…”

“…20 One study demonstrated the presence of significant amounts of amplifiable DNA in expired plasma, which can potentially contaminate CB samples prepared with PT. 14 Theoretically, cell-free DNA and circulating tumor DNA may interfere with molecular tests. Therefore, further analysis of the safety of the PT method for molecular tests using CBs is needed.…”

“…However, their protocols are often cumbersome and frequent low cellular yield is the leading cause of dissatisfaction 13 . Other problems may also occur, such as potential DNA contamination in the PT method, 14 and poor stability of egg white properties in the egg albumen method. In the collodion bag method, the procedure should be performed in a fume hood because the collodion solution contains ethanol and ether.…”

Cell block practices in European cytopathology laboratories

“…First, plasma contains cell-free DNA, 10 which might be a source of DNA contamination and can lead to a false-positive or false-negative results of molecular analysis. 11 Moreover, thrombin reagents might contain contaminant extraneous cells, 7 and this could also interfere with the results of molecular or immunocytochemical analysis. 7,10 Second, sources of plasma and thrombin are not standardized and vary not only between laboratories but also in each individual laboratory over time.…”

“…However, there are several reasons why this method should no longer be used in a modern cytology practice. First, plasma contains cell‐free DNA, 10 which might be a source of DNA contamination and can lead to a false‐positive or false‐negative results of molecular analysis 11 . Moreover, thrombin reagents might contain contaminant extraneous cells, 7 and this could also interfere with the results of molecular or immunocytochemical analysis 7,10 .…”