Special edition: Interview with DBS extraction expert Donald H. Chace, PhD
In this special instalment of our dried blood spot (DBS) blog series, we speak with Capitainer’s Senior Application and Product Specialist, Donald H. Chace, PhD, MSFS, FAACC, to learn more about the important considerations surrounding sample recovery and DBS extraction . Dr. Chace is an expert in mass spectrometry and in the analysis of DBS samples, and he is one of the primary developers of the newborn metabolic screening test known as Tandem Mass Spectrometry of Amino Acids and Acylcarnitines.
Dried blood spot (DBS) testing is a microsampling method based on dried spots of whole blood, which has been used for quantitative or semi-quantitative analysis of hundreds of molecules involved in health and disease. DBS methodology arose from work carried out in the 1960s by American microbiologist Robert Guthrie, who wanted to develop new testing methods for the rare metabolic disorder phenylketonuria (PKU).
Guthrie, being a microbiologist, developed a microbiological-based assay known as a bacterial inhibition assay, to detect PKU. Here, bacteria that could not grow without phenylalanine in the culture media were added to an agar plate that also contained the chemical β-2-Thienylalanine. β-2-Thienylalanine is an analogue of phenylalanine, which inhibits bacterial growth by inactivating proteins. A Guthrie card that contained a DBS from an infant heel prick was then placed on to the agar. Guthrie discovered that phenylalanine reversed the inhibitory effects of β-2-Thienylalanine, so that if the tested blood contained phenylalanine, the bacteria grew around the blood spot, and this indicated that the patient had PKU.
Because Guthrie’s assay was agar-based, a blood spot was a perfect sample format in addition to being a format that could be easily collected, safely transported and tested for the analysis of metabolites associated with PKU and subsequently a few additional disorders. Many new diseases and technologies for their detection followed Guthrie’s pioneering work in newborn screening (NBS).
- Donald, before we get into the details, can you describe the components in a dried blood spot?
Sure. A DBS has two components – the first component is the filter paper that the blood is added to, and the second component is the dehydrated components of whole blood within the filter.
For standard NBS, whole blood is added to a filter paper that comprises a cotton fibre matrix. When added to this matrix, the lipid membranes of blood cells will lyse and release their contents into plasma. Both plasma and cellular components (e.g. membrane lipids, intracellular contents) will mix together and distribute throughout the filter in a manner similar to adding water to a sponge. The cotton fibres of the filter, called linter, are made from cellulose. The fibres form a complex grid, creating channels and spaces for both large and small molecules, such as peptides and proteins, DNA, RNA and other nucleic acids, minerals, metabolites and other small molecules.
Because blood contains water, it will form hydrogen bonds with the cellulose. This causes the fibres to expand, and small and large molecules will form hydrogen bonds with the cellulose and water, with the largest and smallest molecules residing in the largest or smallest spaces, respectively.
Choosing a DBS extraction method and the importance of volume control
- What are the main approaches to recovering a DBS from the filter?
There are two main approaches to DBS extraction, the choice of which will depend on the analyte(s) of interest. One can either release everything from the filter by adding enough water to the DBS to reverse all the hydrogen bonds, or one can add an organic solvent to ‘clean up’ the filter.
With water-based DBS extraction, the molecules and filter paper become hydrated which causes expansion of the fibres, and the channels open up. Water replaces the hydrogen bonds to the paper, to release molecules and proteins. Sometimes, buffers with different pHs are used here to assist in the release of large and highly polar molecules in as small a volume as possible. It is important to bear in mind that recovered blood is, at best, haemolysed blood with no ability to separate cells from plasma. Subsequent analysis of such a sample will be similar to analysing diluted blood because recovery will by necessity require a larger volume than the initial blood volume added to the filter paper.
The other approach, which involves the use of organic solvents, can aid in the selective recovery of molecules of interest for downstream analysis. Organic solvents such as methanol or acetonitrile will denature proteins and large molecules but not metabolites. Adding such solvents to the filter paper can be thought of as a clean-up step where only the least polar compounds and small molecules are removed. This extraction approach is especially suited for metabolite analysis using mass spectrometry. There are many combinations of solvents, and these can be chosen to match the polarity of the analytes of interest.
In NBS there are a variety of methods and platforms for DBS extraction. Some require aqueous solutions while others require organic solvents. In any case, choosing the recovery approach requires some prior knowledge about the analytes of interest, so it is advisable to check the literature in advance to see if others have extracted your chosen analyte(s) from a DBS, or at least from liquid blood.
- Some researchers have reported using physical methods to recover samples from DBS filters, such as squeezing, sandwich vacuum, and bead beating. Do these methods impact sample integrity in any way?
There is no one-size-fits all answer here. The physical methods you mentioned may not directly affect the integrity of small molecules or other markers; however they might impact the sample if they generate heat or cause other by-products of action. For example, excessive shaking during bead beating or heat generated during sonication could denature proteins in the sample, and this is a major problem if proteins are being analysed.
In general, physical methods should not be the first choice when proteins in the sample are of interest. If physical methods are used, they should be carefully validated as with any other laboratory method, to confirm that they don’t negatively impact sample integrity.
- Let’s talk about the impact of recovery volume on efficiency. For instance, if we want to measure an analyte that we expect to be present in very low amounts in a DBS, what tricks can we use to get the sample out without it becoming too dilute?
This is a hard question to answer and frankly more innovation is needed. Based on my experience in NBS, I can share a rule of thumb that seems to work well. For each 6 mm punch or disc, we generally use 200 μL of solvent for DBS extraction. A regular shallow microtitre well can usually handle up to 300 μL of liquid (it can accommodate more but there is risk of splashing and cross contamination), so the solvent volume can be adjusted according to the size of the punch, which may be 3, 6 or even 9 mm in diameter. In any case, the blood volumes will likely be anywhere from 3-10 μL per punch.
But no matter what you do, a sample that has been extracted from a disc will be more dilute than a liquid blood sample on a volume-to-volume basis. Using lower volumes of water or solvent for DBS extraction will result in less than 100% of the material being recovered. In addition, there will always be some residual fluid left over in the disc. There are some methods that may help in minimising this loss, for example, forcing the liquid out of the disc through compression, or vacuuming the filter between two plates where the plate with the paper and residual fluid is forced against a clean plate. A much more favourable alternative may be to add an internal standard to the disc to begin with. This will allow you to control for losses as long as the downstream assays are sensitive enough – stay tuned for more on that approach!
Generally, though, the analytical platforms available for DBS analysis will determine the limits of detection and the types of samples that can be presented to it. There must be enough of the molecule of interest in the sample to be detectable by the platform in question. For instance, an extraction method with a low recovery rate might not yield enough metabolites for a mass spectrometry assay. On the other hand, switching to an assay format that offers greater sensitivity, e.g., certain immunoassays, might be a valid consideration, although immunoassays have historically been prone to selectivity limitations. In practice, the extraction method and analytical platform will need to be validated on a case-by-case basis for each analyte of interest.
Cotton-based DBS filters and pre-cut discs are highly advantageous
- You mentioned that cotton-based filter papers are widely used in NBS. What are the advantages of using cotton?
The major advantages of using cotton are that the cotton fibres, i.e. the linter, create a mesh as they begin to dry and this allows the matrix to expand or contract in the presence of water, much like a sponge. This is very important because it is the swelling and then recompression of the filter paper that enables molecules within the blood to first float around and associate in all the nooks and crannies when spotted to the filter, and then when drying starts the fibres form around these molecules, stabilising them. This physical attribute of cotton is much more favourable than a rigid membrane. As a side note, as the water is removed from the filter paper by drying, proteins in the sample will become enzymatically inactive, and molecules in a DBS are generally more stable without the presence of water.
All of Capitainer’s DBS sampling devices contain pre-cut filter discs made of cellulose that is similar to that used in NBS.
- What are the important considerations in relation to the filter discs that are used in downstream analysis?
The classic DBS cards (the so-called Guthrie cards) used in NBS are large sheets of paper that will absorb as little or as much blood as is applied. However, how much blood is present in a spot will vary depending on the thickness of the paper and the volume of blood applied. Blood will penetrate the spot but also run laterally spreading like water on a floor. Because lateral and vertical penetration rates are not the same, the blood volume will vary in discs of a given diameter punch. For this reason, Guthrie cards have to follow an exact standard for paper thickness, absorption and a target area, which is depicted as a dashed circle on the card. This is the only way to control the volume when using classic DBS cards. Ultimately you have to punch a disc from the card, and you have to know what the volume is in that disc, based on the standard.
Pre-cut discs are more convenient for lab handling than Guthrie cards, and they also allow the user to avoid any contamination that may be introduced by a card puncher. With a pre-cut disc you collect the entire applied volume on the paper, so assuming you know what that volume is, you will be able to calculate exactly what is in the punch to a much higher degree of precision than with classic DBS cards. However, the downside of a pre-cut disc is that it limits the volume of blood that can be absorbed.
Finding a DBS extraction method for your analytes of interest
- What advice can you give to someone seeking an extraction method for a given analyte? Is it possible to apply a published method?
Generally, other published DBS extraction methods for the analyte of interest will be a good start, provided that the method has been performed on the same platform. For example, an ELISA-based method to detect a given metabolite will not be useful if that same metabolite is to be detected using mass spectrometry, because of differences in the eluates used to prepare samples for each method.
If the analytical platforms are the same, then the issue of dilution will matter. Sometimes a platform is made for large fully concentrated volumes of blood, and this won’t suit DBS samples without modification. For large long-term projects, one way around this could be to work with the vendor to make modifications to the platform to facilitate low-volume and/or dilute specimens. These were the approaches that my colleagues and I used when validating amino acids analysis in NBS using tandem mass spectrometry.
- Are there types of blood-borne analytes that cannot be measured using DBS, and why not?
There are no real limits to what can theoretically be measured in a DBS unless intact cells are needed, or if one wanted to distinguish between the concentration of a given analyte in red blood cells and in plasma. Beyond those examples, there are a few diagnostic tests in use that absolutely require blood in its original liquid state, but otherwise there are really not that many limitations for a DSB with today’s technology. There are devices that can sample plasma only.
- What are the main pitfalls that one has to watch out for when recovering DBS samples?
Firstly, one must understand the level of precision and accuracy offered by the chosen method. Classic DBS cards are prone to be imprecise unless strict criteria concerning paper thickness, absorption and a target area are applied. But even at that, no conventional DBS card can achieve known precise volumes.
It is also important to consider the properties of the analytes of interest when setting up the workflow from sampling to extraction. For example, when analysing an analyte that is subject to denaturation (e.g. an enzyme), more care will be needed from sample shipment to extraction than a simple organic extract of an amino acid. A related potential pitfall may apply when analysing multiple analytes, because you will have to adopt a method that is generally good for all but not necessarily the optimum for each.
Perhaps not a pitfall, but it’s highly advisable to implement good quality control and quality assurance procedures. This should include validated methods to test the efficiency of sample recovery, especially if developing a new recovery method. It is also wise to include control blood samples that are spiked in the lab with known concentrations of the analyte of interest covering a low, medium and high concentration range. Another approach I mentioned earlier is the addition of an internal standard to every DBS card/disk so that measured analyte concentrations can be normalised to the internal control every time.
Reproducibility and limits of detection are other important aspects for quantitative DBS testing, but they are probably beyond the scope of this discussion.
- Please tell us the specific advantages of Captainer qDBS technology.
First and foremost, Captainer’s devices are highly precise. The qDBS technology is based on capillary microchannels that deliver a precise volume of sample to a pre-cut paper disc, where that entire disc is used in downstream analysis. Over- and under-sampling are nearly impossible with Capitainer’s qDBS technology, which greatly reduces the risk of false positive or false negative results.
Another major advantage of Capitainer’s devices is sample protection, which is especially attractive for home and remote sampling which require that collected samples are transported to a suitable testing lab. Captainer’s DBS cards are sealed until use, which allows them to be dried in a protected manner until received in a lab, and which minimises contamination risk.
Capitainer®B 50 is the Capitainer® blood sampling version for a larger blood sample volume, with two metering channels of 50µL.
- Thank you, Donald, for sharing these expert insights with us. We look forward to hearing from you again.
Get in touch!
We hope that you found this post useful, and that it answered some of your technical questions related to DBS extraction. If you have a question about a technical aspect not covered here, or if you have a comment to part of this discussion, please do get in touch with us at:
support@capitainer.se,
or Contact Dr. Chace directly at:
donald.chace@capitainer.se
