Comparison of cobas EGFR Mutation Test v2 and PANAMutyper-R-EGFR for Detection and Semi-Quantification of Epidermal Growth Factor Receptor Mutations in Plasma and Pleural Effusion Supernatant
2019; 39(5): 478-487
Ann Lab Med 2022; 42(2): 141-149
Published online March 1, 2022 https://doi.org/10.3343/alm.2022.42.2.141
Copyright © Korean Society for Laboratory Medicine.
1Department of Laboratory Medicine, Yonsei University College of Medicine, Seoul, Korea; 2Department of Laboratory Medicine and Genetics, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea
Correspondence to: Kyung-A Lee, M.D., Ph.D.
Department of Laboratory Medicine, Yonsei University College of Medicine, 211 Eonju-ro, Gangnam-gu, Seoul 06273, Korea
Tel: +82 2-2019-3531
Fax: +82 2-2019-4822
Yoonjung Kim, M.D., Ph.D.
Department of Laboratory Medicine, Yonsei University College of Medicine, 211 Eonju-ro, Gangnam-gu, Seoul 06273, Korea
*These authors contributed equally to this work.
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Standardization of cell-free DNA (cfDNA) testing processes is necessary to obtain clinically reliable results. The pre-analytical phase of cfDNA testing greatly influences the results because of the low proportion and stability of circulating tumor DNA (ctDNA). In this review, we provide evidence-based clinical practice guidelines for pre-analytical phase procedures of plasma epidermal growth factor receptor gene (EGFR) variant testing. Specific recommendations for pre-analytical procedures were proposed based on evidence from the literature and our experimental data. Standardization of pre-analytical procedures can improve the analytical performance of cfDNA testing.
Keywords: Cell-free nucleic acid, Circulating tumor DNA, Pre-analytical phase, Epidermal growth factor receptor, Clinical practice guidelines
Epidermal growth factor receptor gene (
In addition to the differences in test platforms, the pre-analytical phase of cfDNA testing largely influences the test results . Optimization of ctDNA testing is challenging because of the very low proportion of ctDNA in the background wild-type cfDNA and a high susceptibility of short-fragmented cfDNA to degradation [6, 7].
To achieve optimal performance of plasma
The survey was conducted between December 2018 and January 2019 in 19 Korean clinical laboratories performing or referring for plasma
The results of this survey are summarized in Tables 1 and 2. Specifically, 63.2% of laboratories conduct the test in-house, while 36.8% of laboratories send the samples to other laboratories for testing. The average
To develop clinical practice guidelines, we performed a comprehensive literature search in PubMed, KoreaMed, and Google Scholar for articles in English or Korean related to the pre-analytical phase of
The pre-analytical procedures involved eight steps. Specific recommendations for each step were discussed using evidence from the literature and our experimental data. The recommendations are summarized in Table 3.
Cellular DNA is released from lysed white blood cells (WBCs) during the clotting process in serum collection tubes. Consequentially, cellular DNA concentrations are higher in the serum than in the plasma, which may lead to cfDNA becoming diluted with cellular DNA in the serum. Therefore, plasma is a more suitable sample than serum for cfDNA analysis [6, 8, 9].
Tubes with or without cell stabilizer can be used for blood sample collection. For tubes without cell stabilizer, an EDTA tube is recommended and is preferred over other anticoagulants because of the small increase in total DNA when plasma separation is delayed [6, 9–11]. cfDNA analysis using EDTA tubes has been validated previously [6, 11, 12]. It is recommended that sample processing be performed immediately after collection. WBCs are more stable in tubes containing a cell stabilizer, such as the Cell-Free DNA BCT (Streck, La Vista, NE, USA), Cell-Free DNA Collection Tube (Roche Diagnostics), PAXgene Blood ccfDNA Tubes (Qiagen), and Dxtube (Dxome, Seoul, Korea). Thus, if the whole blood cannot be processed within 4–6 hours after collection, tubes with cell stabilizer should be considered . For details on the storage conditions of whole blood before plasma isolation, refer to the section on ‘Storage requirements for whole blood’.
In general, the extracted DNA concentration is increased as the plasma input volume is increased [14, 15]. However, over- or under-filling of the blood volume specified by the manufacturer can cause inaccurate test results by altering the blood and additive ratio . For Roche’s Cell-Free DNA Collection Tube, Qiagen’s PAXgene Blood ccfDNA Tubes, and Dxome Dxtube, the manufacturers suggest 8.5, 10, and 9 mL of whole blood as the optimal sampling volumes, respectively.
Following blood collection, the tubes should be gently inverted 5–8 times immediately to ensure proper mixing of the anticoagulant with the blood. Delayed mixing of blood and anticoagulant can cause the blood to clot and release cellular DNA from the WBCs. Shaking of the tubes should be avoided to prevent hemolysis.
Following collection, whole blood samples should be transported carefully within the stipulated time frame and with minimal movement and temperature changes as per the sample storage requirement. Agitation of the samples should be avoided to prevent hemolysis and cellular damage, which can lead to cellular DNA release [9, 13].
For EDTA tubes, whole blood should be processed according to the manufacturers’ instructions. If there are no specific instructions, whole blood in EDTA tubes should generally be processed within 4–6 hours at room temperature (18°C to 25°C) or 4°C. The whole blood in EDTA tubes should be processed as soon as possible because cellular DNA is released from nucleated cells over time. Previous studies revealed a remarkable increase in the total DNA concentration at 4–6 hours after blood collection [6, 9, 10, 17–21]. Although there is no remarkable difference in sample stability between room temperature and 4°C for a short duration, samples tend to be more stable at 4°C than at room temperature when blood processing is delayed [8, 9, 17, 22].
For tubes containing cell stabilizer, whole blood should be stored according to the manufacturers’ instructions. Generally, blood samples in tubes with cell stabilizer should be processed within 7–14 days at room temperature. For Cell-Free DNA BCT, the manufacturer suggests a stable storage duration at a temperature of 6–37°C for 14 days, while for the Roche Cell-Free DNA Collection Tube and Dxtube, the manufacturers suggest a stable storage period of seven days at 18–25°C. For PAXgene Blood ccfDNA Tubes, the manufacturer indicates that samples are stable at room temperature (15–25°C) for up to seven days or at higher temperatures (up to 35°C) for up to one day. In previous studies, there was no remarkable difference in sample stability and variant detectability between various commercial tubes with cell stabilizer [23, 24]. In general, it is recommended that plasma is separated as soon as possible after blood is drawn, even when using tubes containing cell stabilizer.
For tubes containing cell stabilizer, whole blood should be processed according to the manufacturers’ instructions. The centrifugation conditions proposed by the manufacturers are as follows. For Cell-Free DNA BCT, two separate centrifugation conditions are recommended: 1) first centrifugation at 300 ×
As described above, single or double centrifugation is recommended by different manufacturers. Generally, if there are no instructions for tubes containing cell stabilizer or those with EDTA, double centrifugation is recommended to produce cell-free plasma . In single centrifugation, cfDNA can be diluted by cellular DNA from remnant nucleated cells in the plasma.
To compare cfDNA yields and cellular DNA contamination between double and single centrifugations, 20 mL peripheral blood was collected into an EDTA blood collection tube (Vacutainer K2EDTA) (BD Biosciences, Franklin Lakes, NJ, USA). Each 10 mL volume was then split into 2×5-mL aliquots and then incubated at room temperature for two hours. Plasma was isolated under the following two conditions: Centrifugation_Condition_1: first centrifugation at 1,600 ×
Short-length DNA (cfDNA) was more abundant when using Centrifugation_Condition_2 (with a second high-spin centrifugation at 16,000 ×
As cfDNA degradation by nucleases can continue after plasma separation, cfDNA should be extracted immediately from the separated plasma . If the test is not performed immediately, the plasma can be stored for up to three hours at 4°C . For long-term storage, plasma should be frozen at −20°C or −80°C . Because freezing and thawing of plasma cannot be performed more than once, samples should be aliquoted into multiple tubes for subsequent testing .
The length of cfDNA is shorter than that of genomic DNA [29, 30]. Therefore, cfDNA should be extracted using a method that targets low-molecular-weight DNA. It is recommended to use the cfDNA extraction kit specified in the cell stabilization tubes or
To quantify cfDNA, spectrophotometry (e.g. NanoDrop), fluorometry (e.g. Qubit), real-time PCR, and digital PCR methods can be used . To quantify low concentrations of cfDNA, spectrophotometry is less accurate than fluorometry [15, 35]. Automatic electrophoresis systems, such as the Bioanalyzer and TapeStation, allow quantification and size measurements of cfDNA.
Storage conditions for cfDNA should follow the recommendations by the cfDNA isolation kit manufacturer. For the Cobas cfDNA Sample Preparation Kit, extracted cfDNA should be used within the following recommended storage periods: −15°C to −25°C for up to two freeze thaws over 60 days, 2–8°C for up to 21 days, and 15°C or 30°C for up to seven days. For the QIAamp Circulating Nucleic Acid Kit, the recommended storage conditions for eluted cfDNA are 2–8°C for 24 hours and −15°C to −30°C for longer than 24 hours. For the QIAsymphony Blood ccfDNA Kit, the extracted cfDNA is stable at 2–8°C for up to one month and −20°C or −80°C for long-term storage and up to three freeze-thaw cycles. For the MagMAX Cell-Free DNA Isolation Kit, purified cfDNA can be stored on ice for up to 24 hours and at −20°C for long-term storage.
If there is no recommendation, downstream analysis should be performed immediately. cfDNA should be stored below −20°C. Repeated freeze–thaw cycles can accelerate DNA degradation; thus, multiple aliquots are recommended for subsequent testing. Studies have shown that up to three freeze–thaw cycles of cfDNA do not significantly affect the DNA stability [9, 20].
This survey was conducted when plasma
The T790M variant-positive rate in the plasma of patients who develop progressive disease from prior EGFR-TKI therapy has been reported to be 17.5%–48.1% depending on the testing methods [36–38]. Although we did not divide the T790M variant-positive rate according to the previous history of EGFR-TKI therapy, the results in a high proportion (73.7%) of responders revealed that the actual T790M variant-positive rate was lower than expected. Various factors may affect the T790M variant-positive rate, including patient factors, test sensitivity, and limited criteria of health insurance. Currently, in Korea, plasma
Further, pre-analytical factors, such as transport time, plasma volume, centrifugation protocol, and sample storage, would account for a decreased T790M variant-positive rate. Seven of the 19 (36.8%) institutions answered that they send samples for plasma
In conclusion, these clinical practice guidelines will serve to standardize the pre-analytical procedures of plasma
Shin S and Woo HI reviewed the literature and wrote the original draft. Kim JW provided advice. Kim Y and Lee KA designed and supervised the study and reviewed and edited the manuscript.
No potential conflicts of interest relevant to this article are reported.
This study was supported by the Research Fund of Quality Control Committee, Korean Society for Laboratory Medicine (KSLM Research Project 2020-01-006) and Korean Society for Genetic Diagnostics.