Automatable Processes and Future Directions in Dried Blood Spot Analysis

The application of dried blood spots (DBS) for the storage, shipment, and quantification of xenobiotics has been gaining popularity in recent years.1–8 The invention of this technology can be attributed to Guthrie in the 1960s for an assay he developed for the detection of phenylketonuria.9 This procedure of collecting blood on specialty paper was further used in population screening of newborns for the detection of a variety of inherited metabolic diseases.

While application of this technique for use in the pharmaceutical industry may be in its infancy, great strides have been made by GlaxoSmithKline (GSK, King of Prussia, PA) and several other companies to adopt the method for routine, small-molecule quantification. The ethical and cost benefits are in the areas of sample collection and storage. The technology can help reduce, refine, and replace (3Rs) the use of animals in drug development. The challenge is the ability to collect adequate samples while staying within the acceptable total blood collection volumes and avoiding excess animal use. The use of DBS for pharmacokinetic (PK) and toxicokinetic (TK) studies is aptly suited, considering that a typical blood spot uses only 15 µL of blood. This small collection volume also permits serial sampling of one rodent rather than composite bleeds from several animals, as well as pediatric studies. Another consideration is the cost savings in regard to shipping and storage. Because neither dry ice nor refrigeration is required, shipping bulk is minimized and clinical trials can be performed in remote areas.

Typical analytical procedures for the use of DBS include the following: Blood is spotted onto specialty paper (typically 15–20 µL, but some researchers use up to 100 µL), and the spotted cards are placed in a vertical drying rack for a period of 2 hr to overnight. After drying, the cards are sealed in a plastic zipper storage bag with a package of desiccant, which can be transported to the analytical laboratory under ambient conditions. In the laboratory, the card is punched (typically 3–4 mm) and placed into a 96-well format. The extraction solvent is added and vortex-mixed for 30–60 min, and the supernatant is then removed and injected into an LC-MS-MS.

While the benefits of this technology are clear for collection and storage, bioanalysis and assay development prove to be more complex than a standard plasma assay. Optimization steps include card type and extraction solvent selection. Other optimization steps, dependent on required lower limit of quantification (LLQ), may include spot and punch size. Four commercially available card types are currently used for the development and validation of DBS assays. Two of the cards are chemically treated with additives to lyse cells (antibacterial) and denature protein, while the other two cards are untreated. While these card additives have been shown to increase on-card stability in certain instances, they also increase suppression in MS detectors and require adequate LC separation, thereby adding to method development time.

DBS card format

Figure 1 - DBS card formats. From left to right: DMPK-C (GE Healthcare, Piscataway, NJ), 226 (Ahlstrom, Helsinki, Finland), Media (Varian, Palo Alto, CA), FTA® (DMPK-A) (GE  Healthcare), and FTA Elute (DMPK-B) (GE Healthcare).

Figure 2 - Dimensions of a standard, automatable DBS card.

Figure 1 shows currently available DBS card formats. The cards contain filter paper or similar media surrounded by a cardboard stock to simplify handling and manipulation. They are also color-coded to ease identification. The standard dimensions, as shown in Figure 2, are designed for ease in drying, storage, shipment, and automation. As can be seen in Figure 2, a typical card contains designated areas for up to four spots and ample room for placement of a bar-code identification label. Investigation is under way to design a plastic-coated card that would more adequately protect the DBS during shipment and storage. Disadvantages with the current plastic-coated design include increased drying time associated with the cover and added manual manipulation prior to punching and processing the card. 

Blood spotting and storage

In preclinical environments within GSK, tail pricks are the preferred mode of bleeding for rodent models; however, other sampling techniques exist. Blood samples can be placed into microvessels for collection, followed by spotting with graduated EDTA-coated capillaries. These capillary tubes can also be used to collect the blood from the tail pricks, followed by direct spotting onto the DBS media. Sampling from larger animals involves collecting blood into a microvessel, followed by spotting using capillary tubes or pipets. Experienced safety assessment personnel at GSK are capable of holding multiple capillary tubes simultaneously to speed up the process. The Culex, an automatic blood collection device from BASi Inc. (West Lafayette, IN), is also being investigated for collecting blood from preclinical species.

In clinical studies, finger pricks are the preferred mode of bleeding9 when compared in venous sampling. As with preclinical DBS collection, blood can be collected into a microvessel or spotted directly with a capillary tube. In the bioanalytical laboratory, repeater pipets are the preferred route of spotting on DBS cards due to the larger volume of blood prepared and the number of cards being spotted.

Due to the low volume of blood being spotted onto the DBS media, extensive experiments have been performed to investigate the effect of blood spot volume variation in respect to assay accuracy and precision. In all assay validation at GSK, a ruggedness experiment is performed to account for spot volume variation that may occur. Blood is spotted in volumes ranging from 10 to 20 µL, and the spots are compared to each other. Based on homogeneity in compound distribution within the DBS spot, and a fixed-volume punch, all values are normalized and accuracy is within 15%.