We developed the direct cfDNA quantitation method based on our SuperbDNA™ Technology. The method uses microliters of plasma and detects 0.39 ng/ml of cfDNA using the luminometer (VznHealth™ Direct cfDNA Test ) and 0.09 ng/ml cfDNA using Luminex MagPix (QuantiDNA™ Direct cfDNA Test) after plasma dilution. The two platforms show consistent quantifications of cfDNA (see support data below).
Luminometer Detection
Luminex MAGPIX Detection
Suggested Workflow for cfDNA Quantification for Real-Time cfDNA Level Monitoring
As we reviewed above, cfDNA has been widely studied for
Comparison of healthy individuals and various types of patients
Disease stage and treatment monitoring for a certain type of patients
If the cfDNA does not need to be purified for further mutation or epigenetic change detection,
The workflow for cfDNA quantitation is summarized below. At different time of points at disease progression or therapy, cfDNA can be quantitated using the direct cfDNA quantitation kits. However, it is not always necessary to extract cfDNA for the quantitation purpose if no cfDNA changes need to be detected at different time points. Often, the cfDNA qualitative analysis is necessary for diagnosis of diseases for particular genetic changes or therapy guidance based on certain gene mutations, such as drug resistance development after use of first or second generation of tyrosine kinase inhibitors (TKIs). Below figure shows the suggested workflow for cfDNA monitoring for disease progression or therapy.
Personalized Medicine and Precision Medicine
The future trend for healthcare is personalized medicine (tailoring of medical treatment based on
Liquid Biopsy as a Tool for Precision Medicine
As a personalized medicine tool,
Rapid growth in liquid biopsy analysis, especially analysis of circulating cell-free DNA (cfDNA) provides valuable information in disease diagnosis and therapeutics: early diagnosis, risk assessment for metastatic relapse or metastatic progression, patients stratification, disease real-time monitoring during therapies, identification of somatic mutations for different therapeutic targets, drug sensitivity and resistance understanding, and disease progression such as metastasis development, and many others.
What is cfDNA?
cfDNA is commonly found circulating in plasma. First discovered in plasma in 1948 by Mandel and
It is thought that cfDNA comes from apoptosis (programmed cell death) and cell necrosis. The size of the cfDNA varies depending on the source. It is reported that cfDNA from apoptosis can be 100 bp to 500 bp, average about 170 to 180 bp while the cfDNA from necrosis is usually much bigger and can reach more than 1 kb.
What is the Correlation between the Levels of cfDNA and Diseases?
The publications for cfDNA from plasma have been increasing over the years from <100 in 2007 to >300 in 2016. cfDNA has been studied as a potential biomarker for different diseases including, but not limited to cancer, cardiovascular diseases (sepsis, myocardial infarction), hemodialysis, inflammation, infection, ischemic stroke, and connective tissue diseases, pregnancy-associated disorders and transplantation rejection.
Quantitative cfDNA Analysis: Lack of Standardization
cfDNA quantification for various diseases including cancer, cardiovascular, infection and autoimmune diseases and their treatment indicate that the cfDNA levels are significantly higher in patients with these diseases than in healthy individuals. However, lack of standardization in cfDNA quantitation makes comparison of different studies difficult. In most of the studies, cfDNA is extracted and purified first, followed by assessment. This adds variation to the cfDNA quantitation. Although cfDNA is a potential biomarker for disease prediction, diagnosis and prognosis, only limited studies have firm conclusions. Standardization of cfDNA quantitation method will be critical for future correlation studies of cfDNA quantitation with different diseases and demonstration of cfDNA used as a potential disease biomarker.
Quantitation Methods of cfDNA
Although many publications quantify cfDNA for different diseases studies, the lack of a standard for quantitation of cfDNA has generated different cfDNA results, especially for the methods that involved in different DNA extraction methods and following assessment and calculation. The cfDNA quantitation method can be classified as direct quantitation and indirect quantitation methods. Direct quantitation of cfDNA directly measures cfDNA concentration without the need for DNA extraction/purification, such as direct PCR, direct SYBR Gold assay, or
Below figure shows the comparison of direct and indirect DNA quantification methods.
Compared to indirect quantification, the advantage of the using direct quantitation methods include:
The cfDNA extraction and purification is not necessary for determination of the concentration
cfDNA concentration measurement needs minimum of plasma rather large amount of plasma
Reagents used in DNA extraction may affects the quantitation results in the following procedures
The procedure minimizes loss of cfDNA during cfDNA purification
The procedure saves hands-on time and may get adapted to automation easily for high throughput needs
Supporting Data – Assay performance for VznHealth™ and QuantiDNA™ Direct cfDNA Test
| VznHealth™ Direct cfDNA Test | QuantiDNA™ Direct cfDNA test | |
|---|---|---|
| Detection Limit | 0.39 ng/ml (7.8 pg in 20 µl assay) | 0.09 ng/ml (1.8 pg in 20 µl assay) |
| Quantitation Range | 0.39 to 50 ng/ml | 0.09 to 25 ng/ml |
| Intra-Assay Reproducibility | <15% | <10% |
| Inter-Assay Reproducibility | <15% | <15% |
| Interference from Hemoglobin | No | No |
| Interference from cholesterol | No | No |
| Interference from EDTA | No | No |
Both VznHealthTM Direct cfDNA Test (plate-based) and QuantiDNATM Direct cfDNA (bead-based) test are used for measurement of cfDNA concentration change for one prostate cancer patient after 1 to 5 days of radiation treatment versus cfDNA levels before treatment (in absolute number or expression as fold change).
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