According to the Centers for Medicare and Medicaid Services (CMS), Clinical Laboratory Improvement Amendment (CLIA) registration is required for entities that perform a single test on, “materials derived from the human body for the purpose of providing information for the diagnosis, prevention or treatment of any disease or impairment of, or the assessment of the health of, human beings”.

To date, only two next generation sequencing (NGS) instruments/tests have been approved or cleared by the FDA. All other NGS based tests are developed in house as laboratory developed tests (LDTs), and are regulated under CLIA. CLIA regulations are required to certify the validity of a test. Validity is established by measuring:

1. Accuracy
2. Precision
3. Analytical sensitivity and specificity
4. Reportable reference range or interval

For next generation sequencing tests this means several sequencing based metrics are required:

In addition to CLIA, the College of American Pathologists (CAP) has several specific guidelines for NGS labs. These include consideration for validated sample extraction, library preparation, barcoding, pooling and target enrichment. Each protocol has specific quality metrics associated with it. In addition to the wet lab, bioinformatics pipelines must be validated and tested for how precise and sensitive variants are called.

Clinical regulation of NGS based tests are undergoing rapid change as new NGS tests enter the clinic, and older ones are improved. As these changes happen, both CAP and CLIA requirements for NGS are updated on a yearly basis.

The most common NGS based assays or tests performed in a CLIA/CAP setting today include:

1. Exome sequencing
2. NGS gene panel sequencing
3. Whole genome sequencing
4. Cell free DNA sequencing
5. Metagenomic sequencing

Genohub has existing relationships with 7 service providers offering nucleic acid extraction, library preparation, sequencing and data analysis under CLIA and CAP. To obtain NGS services under CLIA/CAP accreditation, submit a request here: https://genohub.com/ngs.

Identification of biomarkers that indicate presence of disease are highly sought after. Non-invasive methods to measure those biomarkers are even more valuable. By extracting and measuring cell-free DNA, scientists have satisfy both.

Cell free DNA are degraded fragments released in plasma. Elevated levels of cfDNA are found in cancer states, making assessment of somatic genomic alterations from tumors possible using sequencing. Cell free fetal DNA (cffDNA) can be found as early as 7 weeks gestation, and analysis of cffDNA is already being used in non-invasive prenatal diagnostics. Cell free DNA (cfDNA) in blood was first described by Mandel and Metais in 1948 [1] but only recently has been identified as having utility for prenatal testing and disease diagnostics and monitoring.

Unlike mutations that are passed from a parent to child and are in every cell of your body, somatic mutations form during a person’s life. These somatic mutations are present in tumor cell DNA and are an excellent biomarker if they can be measured and monitored.

Acquiring tumor DNA often requires a biopsy, a potentially risky and invasive procedure. In many cases presence of a tumor or the ability to biopsy is not even an option for patient. During tumor turnover and progression, apoptotic and necrotic cells release small pieces of their DNA (cfDNA) into the bloodstream. The amount of cfDNA in the blood steam is influenced by clearance and filtering of the blood and lymphatic circulation.

Detecting cfDNA in plasma is called a ‘liquid biopsy’ and is already a popular method for obtaining clinical samples for prenatal testing, disease diagnostics and monitoring. One of the challenges of liquid biopsies, are standardization of the isolation procedure and maintaining  uniform specificity and sensitivity. Extraction of cfDNA can be carried out using magnetic beads or silica matrices along with chaotrophic salts, such as guanidine thiocyanate. While several commercial approaches (Table 1) exist, none have undergone rigorous large patient scale studies. Once more information is known, universal standardization should allow greater clinical utility.

Commercial kits for extraction of cfDNA need to be designed to extract uniform DNA copies from varying biopsy volumes. Scalability and adaptability for cell free fetal and ctDNA are important considerations. Below we highlight current kits available in the market. In a future blog post we’ll discuss isolation and sequencing standardizations required for broader use of cfDNA liquid biopsy.

Table 1.