Tag: molecular diagnostics

I was privileged to attend this year’s Association for Molecular Pathology (AMP) meeting in Charlotte, North Carolina, in the beginning of November. I really enjoy this meeting – it is relevant to everything our lab does with sessions offered in topics of Hematopathology, Infectious Diseases, Solid Tumors, Inherited Diseases, and just recently added, Bioinformatics.

It is exciting to meet and discuss with others in this field, especially other laboratory technologists. AMP has done a wonderful job of including those of us who perform the bench work, offering discounted memberships, as well as learning opportunities on their website, and even an award especially for technologists’ exemplary posters/abstracts presented at the annual meeting.

This year’s meeting offered the previously mentioned topics, but an emerging trend was evident – testing cell-free DNA (cfDNA) obtained from sources other than tissue biopsies, such as plasma or urine. This quarter’s post will deal with the reason behind this and the technology for testing such specimens, specifically plasma.

Cell-free DNA has become an attractive source for tumor testing recently. This source can be tested when a tissue biopsy is just not possible, such as when a patient has progressed to the point that surgery is not recommended. Here is the biology behind why this can work as a source of tumor DNA:

Figure 1. http://www.intechopen.com/books/methylation-from-dna-rna-and-histones-to-diseases-and-treatment/circulating-methylated-dna-as-biomarkers-for-cancer-detection

The sources of DNA in a sample of whole blood (as shown in Figure 1) are:

• white blood cells
• degraded white blood cells (cfDNA)
• degraded tumor cells (cfDNA)
• circulating tumor cells (CTCs).

Because of the biology of tumor cells, they have higher turnover than other cells in the body. Due to this, a larger fraction of the cfDNA in the plasma is from tumor cells. We can take advantage of this with a so called “liquid biopsy” – with 10 cc’s of whole blood, we can attempt to capture about 10ng of cfDNA and test this for possible resistance mutations to the therapies the patient may be on.

Many of the posters and several of the sessions at the AMP meeting dealt with cfDNA. Several pre-analytical steps were stressed in order to have success with this type of specimen.

1. The whole blood needs to be collected, as any other blood specimen should, with care taken to not lyse any of the cells during collection.
2. The collection tube type varies depending on how much time it will take to centrifuge the specimen to obtain the plasma. If it can be spun within two to four hours, a simple EDTA tube is sufficient. If it cannot be spun within a short time, then another tube with special preservatives is required. A Streck tube has been the tube of choice in these situations, but others are becoming available on the market as the demand increases. These specific tubes offer a greater amount of time to capture the cfDNA without white blood cell lysis becoming an issue. This is important, because as the white blood cells lyse, the plasma is flooded with the patient’s normal cfDNA that will dilute out the tumor cfDNA fraction, making it even more difficult to detect.
3. Centrifugation procedures must be altered. The brake should not be applied when stopping the centrifuge because braking can cause the white blood cells to be sheared, which will, again, flood the plasma sample with normal cfDNA. An initial spin should be performed to obtain the plasma, then an additional spin should be performed before extraction of the DNA.

There are multiple kits available on the market for extraction of cfDNA. Once the DNA is extracted it is suggested to measure the DNA fraction with a method that will display the size of the fragments, such as with a Bioanalyzer. Cell-free DNA is about 160-170bp in size and, with the readout from an instrument such as the Bioanalyzer, one can see the size of the DNA, quantitate it, as well as observe any contamination from genomic DNA (shown by a peak >>170bp in size).

Many types of testing are being performed on this cfDNA fraction such as real time PCR, digital droplet PCR, and next generation sequencing. Whichever platform is used, a validation must be performed to ensure a fairly low level of detection (as low as 0.1% or 0.01%) because, many times, the positive tumor cfDNA allele fraction will be very low due to the normal cfDNA in the plasma.

This method of testing non-invasive specimens from patients is an amazing way to help save possibly very sick people from having to undergo a risky surgery. This is yet another use of a new technique in the ever changing world of Molecular Diagnostics!

 

-Sharleen Rapp, BS, MB (ASCP)^CM is a Molecular Diagnostics Coordinator in the Molecular Diagnostics Laboratory at Nebraska Medicine. 

“Blotting,” in relation to molecular diagnostics, is a term that refers to the process of detecting the presence and quantity of DNA, RNA, or protein in cells. There are three main types of blotting procedures that those in the field should be familiar with: Southern, Northern, and Western. Three additional blotting procedures are termed Southwestern, Eastern, and Far-Eastern. These are also summarized in the table below.

Southern Blot Steps

1. DNA is isolated and cut with restriction enzymes.
2. The DNA fragments are then analyzed by gel electrophoresis and separated by size (see previous blog post on Separation and Detection).
3. Depurination – Gel is soaked in hydrogen chloride (HCl) to remove the purine bases from the sugar-phosphate backbone. This loosens up larger fragments before denaturation.
4. Denaturation – The DNA is denatured by exposing the gel to sodium hydroxide (NaOH). Denaturation breaks the hydrogen bonds that hold the DNA strands together.
5. Blotting – The denatured DNA is transferred to a solid substrate (nitrocellulose) that helps to facilitate probe binding and signal detection.
6. Pre-hybridization – Prevents non-specific binding of the probe to other sites on the membrane surface.
7. The membrane is exposed to the hybridization probe, usually a single DNA fragment with a specific sequence to the target DNA. The probe DNA is labelled either with radioactivity or fluorescent dyes.
   

Importance of the Membrane

Nitrocellulose and nylon membranes are best for smaller sized single stranded DNA fragments. It is compatible with many types of buffers and transfer systems. These membranes work well with protein and nucleic acids.

 

METHODS OF TRANSFER
Capillary Transfer	Ÿ Utilizes capillary movement of the buffer from a soaked paper to the dry paper Ÿ Denatured DNA moves from the gel to the membrane
Electrophoretic Transfer	Ÿ Electric current moves the DNA from the gel to the membrane
Vacuum Transfer	Ÿ The force from suction moves the DNA from the gel to the membrane

 

Northern Blots

Northern blots are used in the laboratory to look at RNA structure and quantity. It’s a powerful method that can measure levels of gene expression, as well as structural abnormalities in RNA.

• Needs to take place in an RNase-free environment.
• The samples are applied directly to an agarose gel.
• The sample is cut out from the gel, soaked in ammonium acetate to remove the denaturant (denaturant is inhibitory to the binding of RNA to nitrocellulose membranes), and stained with acridine orange or ethidium bromide.

Western Blots

Western blots detect proteins and separates them according to their molecular weight or charge

• Run using a polyacrylamide gel with molecular weight standards / markers.
• Utilizes capillary or electrophoretic transfer methods.
• Membrane must be blocked with a solution to prevent binding of the primary antibody probe to the membrane.

 

TYPES OF PROBES
DNA Probes	Southern Blots	Complementary to the target gene
RNA Probes	Northern Blots	Complementary to the target sequence
Protein Probes	Western Blots	Antibodies bind to the target protein

 

SUMMARY OF HYBRIDIZATION TECHNIQUES
Method	Target	Probe	Purpose
Southern Blot	DNA	Nucleic Acid	·      Gene structure
Northern Blot	RNA	Nucleic Acid	·      RNA transcript structure, processing, and gene expression
Western Blot	Protein	Protein	·      Protein processing and gene expression
Southwestern Blot	Protein	DNA	·      DNA binding proteins and gene regulation
Eastern Blot	Protein	Protein	·      Modification to western blot using enzymatic detection ·      Detection of specific agriculturally important proteins
Far-Eastern Blot	Lipids	None	·      Transfer of HPLC-separated lipids to PVDF membranes for analysis by mass spectrometry

 

-LeAnne Noll, BS, MB(ASCP)^CM is a molecular technologist in Wisconsin and was recognized as one of ASCP’s Top Five from the 40 Under Forty Program in 2015.

 

 

 

If you’re involved with molecular diagnostics, then L. J. Lee’s team at the Ohio State University would like your input. They’ve developed a new technology using molecular beacons and would love for lab directors, managers, and bench technologists to answer this short survey.