Dried Blood Spot (DBS) or Dry Plasma Spot (DPS) analysis as applied to forensic analytical chemistry (toxicology) for Drugs of Abuse testing is an emerging science that there has been some recent activity in the peer reviewed literature. This post will examine this emerging technology and evaluate the benefits and the limitations of this assay.

Dried Blood Spot (DBS) or Dry Plasma Spot (DPS) is a recent technology when measured against other long standing forms of analysis on different matrices. It was first popularized by Dr. Robert Guthrie in the early 1960’s. It has been used since then primarily in the neonatal context. It is most frequently used as a screening test right after birth to determine whether or not the child has Phenylketonuria (PKU). If you have had a child in the last 30 or 40 years, this is what they are doing right after birth when they perform a fingerprick or footprick test on your newborn. There is some limited and early research into using this method as a screening tests for HIV. Now, some in the forensic arena have watched the research advancements in this technology and are investigating its possible application to the forensic science world. Some are seeking to prove its validity in terms of a confirmation test in Drugs of Abuse analysis.

What also spurs on this research are the strides being made in the clinical and pharmaceutical field. There is a large push to move in this direction in the clinical arena due to cost savings as well as the analyst and collector being exposed to less bio-hazards. There were a lot of presentations on it that I have noticed in the last several national clinical meetings. In the pharmaceutical industry, there is also a push to get the FDA to more readily accept it in animal studies as it will reduce the numbers of animals needed for a given study. Thus, it will save a lot of money. The whole idea is reduce the amount of blood volume necessary, and save money.

In the peer reviewed research that has been published concerning DBS and DPS, there is some limited data in terms of the correlation to whole blood of plasma blood, and in terms of recovery using this method for qualitative and quantitative analysis. It confirms that this is an emerging field and there are still a lot of questions as to its validity especially as to its possible quantitative use.

In the literature, it is confirmed that at this time, there are large scale limitations to this technology. Just to name a few areas of concern:

• There is a difference in venous blood and peripheral blood sampling (especially when considering PK issues). Typically DBS is not taken from the same spot as WB is taken.
• There is a real concern of Random Sampling Error with only  such a small sample being tested. It is a 3mm diameter punch from a 15uL spot which is equivalent to <3 uL blood.
• There have been noted issues with respect of reliably establishing LOQ for some drugs especially inhaled drugs.
• The choice of the cards and their substrates that the dry blood are placed on can cause recovery issues from one brand to another.
• The chemicals impregnated into FTA DMPK A & B can co-extract with an analyte and cause increased suppression effects.
• The assay validation requirements are up in the air. Currently, there is no guidance from regulators around the world. There are no clinical standards published for its use either.
• The choice of extraction solvent can present issues (acetonitrile gives appreciably “dirter” extracts than methanol, and high proportions of water can give very dirty extracts).
• There are suspected to be storage and transportation issues in terms of the sample card. This is, of course, significant for retest issues and for overall fidelity (stability) of the sample over time. More study is needed (on-card stability experiments).
• Obviously, drying the spot completely on the card is required. Humidity is a factor.
• Where you take the punch from and how big the dried spot is controls whether not the sample is likely to be homogeneous.
• The biggest analytical challenge that has developed over the last year in terms of validating the method is hematocrit (HCT) variability and quantitation issues.
  

High HCT blood, as we would expect, is much more viscous and therefore does not spread out as much on the sample card. High HCT leads to smaller blood spots on the card. Low HCT is the opposite. The issue becomes when you take a fixed punch from the dried blood spot (3 mm diameter), you are sampling a different amount of blood depending upon HCT due to this viscosity issue. With higher HCT, there will be less spread on the card, and therefore, you will be taking more blood. This in turn makes a uniform positive bias (artificially inflated concentration versus the true value).

Even if you expand the punch to sample the entire spot, that does not solve the bias issue in terms of the HCT, it just makes it inversely resulting. For those with lower HCT, there is a uniform bias high when the entire sample on the card is taken for analysis.

When or if this start technology begins to be implemented in the forensic arena, please let me know. I would be most interested in seeing the testimony from a Frye/Daubert hearing on it.