The Need for Standardization in Liquid Biopsy Sample Prep

Reproducible workflows would help clinical labs successfully and routinely perform liquid biopsy assays

Liquid biopsy for cancer patients involves the isolation of circulating tumor cells, circulating tumor DNA, and other tumor-derived materials such as proteins and exosomes from patient blood samples. These non-invasive assays have the potential to revolutionize applications such as screening for cancer, monitoring tumor progression, assessing response to therapeutics, and detecting recurrent disease. Using liquid biopsy, clinicians are able to obtain a more complete picture of tumor heterogeneity and gain information about tumors in locations that would not be accessible by tissue biopsy. 

Despite the advantages of liquid biopsy, the majority of assays still lack evidence of clinical utility and validity, with only four tests obtaining approval from the Food and Drug Administration (FDA). One reason for this is that liquid biopsy assays often lack reproducibility due to the absence of standardization across workflows. 

For clinical labs to successfully implement liquid biopsy, they need to develop easy-to-use, robust, and reproducible workflows that include Standard Operating Procedures across all phases of laboratory testing. Of particular interest is the standardization of pre-analytical workflows for liquid biopsy as assay outcome can be influenced by many different variables during this phase. 

The pre-analytical phase of liquid biopsy

The pre-analytical, or sample preparation, phase of liquid biopsy includes all the steps prior to analysis such as specimen collection, stabilization, transport, enrichment, processing, and isolation and quality assessment of the analyte. The purpose of this workflow is to maintain the integrity of the sample following blood draw and prepare it for analysis. The pre-analytical phase is arguably the most important part of liquid biopsy workflows as 46 to 68 percent of errors occur during this phase. These errors can adversely affect data quality in the following phases and can result in incorrect treatment decisions

A review of the literature reveals many different protocols for isolating liquid biopsy analytes. Researchers have compared different methodological approaches for each step in the sample preparation process. The sheer number of reports combined with the sometimes-contradictory impacts of different pre-analytical variables highlights the urgent need to standardize these procedures. In order to standardize these aspects of the pre-analytical phase, researchers first need to understand their impact on sample integrity and the eventual success of liquid biopsy tests. 

Variables to consider in the pre-analytical phase

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Many different pre-analytical aspects can lead to interlaboratory variability when performing liquid biopsy. These variables include the type of blood collection tubes used, the storage conditions of the blood sample, the time between blood collection and sample processing, the blood processing protocol used, the extraction method used, and the quality assessment method used. The impact of each of these variables depends on the liquid biopsy application. For example, different considerations need to be made when isolating circulating tumor DNA versus circulating tumor cells. 

Of the pre-analytical variables outlined above, the type of blood collection tube used, and the storage conditions of blood samples have been most thoroughly investigated, especially in the context of circulating tumor DNA. Researchers have found that the quality of cell-free DNA  extraction and detection of circulating tumor DNA from patient blood samples is affected by both.

When blood samples destined for circulating tumor DNA analysis are stored in standard EDTA tubes, leukocyte lysis results in an increase in genomic DNA concentration over time, making it more difficult to detect the minute amounts of circulating tumor DNA in the sample. To prevent this increase in genomic DNA, blood samples stored in EDTA tubes that will be analyzed for circulating tumor DNA need to be processed within six hours after the blood draw. 

To overcome the inconvenience caused by this time restriction, there is a growing list of stabilizing reagents and dedicated blood collection tubes designed to preserve cell-free DNA profiles in whole blood. These tubes prevent cell lysis, limiting contamination of the sample with genomic DNA. Blood samples for circulating tumor DNA analysis stored in specialized tubes can be kept at room temperature for a number of days before processing is needed. 

While researchers have made progress in understanding how the type of tube used and storage conditions impact circulating tumor DNA analysis, no consensus on best practices has yet been reached. There are also many other pre-analytical variables whose impacts on circulating tumor DNA analysis are unknown. Information on how these variables impact other applications for liquid biopsy, such as exosome analysis, remain unclear. 

Standardization initiatives

Several international organizations are working toward developing standards for liquid biopsy workflows. These organizations are either working directly to build these standards or are developing the infrastructure needed for data sharing across stakeholders to reach a consensus.

SPIDIA4P is a continuation of SPIDIA, which tackled the standardization and improvement of pre-analytical procedures for in vitro diagnostics. The next phase of the initiative involves working to improve the global health care system by developing selected high-priority pre-analytical European Committee for Standardization (CEN) and International Organization for Standardization (ISO) standard documents. They are also looking to develop corresponding External Quality Assessment (EQA) schemes and implementation tools.

CANCER-ID is another European consortium that is working to establish standard protocols for blood-based biomarkers. They are also working to clinically validate such biomarkers. This consortium is funded by the Innovative Medicines Initiative and is composed of 36 partners from 13 countries.

BloodPAC is an American initiative to accelerate the development, validation, and clinical use of liquid biopsy assays in order to better inform medical decisions so that patient outcomes can be improved. They have developed a collaborative infrastructure that allows for information sharing between stakeholders in the public, industry, academia, and regulatory agencies. They hope that information sharing, and evidence generation will help bring liquid biopsy into routine clinical practice.


Standardization is a critical step in ensuring the clinical utility and validity of liquid biopsy assays. This is especially true for the pre-analytical phase of liquid biopsy workflows, where most errors are likely to occur. While the impact of some pre-analytical variables has been investigated for specific liquid biopsy applications, no consensus has yet been reached on optimal workflows. The efforts by international initiatives such as SPIDIA4P, CANCER-ID, and BloodPAC will hopefully facilitate the standardization of these workflows and usher more liquid biopsy assays into clinical laboratories.

Clinical Lab Manager Cancer Testing Survey

Catherine Crawford-Brown, MSc, MScComm

Catherine Crawford-Brown, MSc, MScComm, is a health science and research writer with a master’s in science communication from Laurentian University. She also has a master’s of science in pathology and molecular medicine from Queen’s University where she worked on developing a liquid biopsy for breast cancer. She was formerly the digital media editor for Lab Manager.