It’s been a standard procedure in obstetrics for 40 years: a doctor or nurse pricks the newborn baby’s heel with a needle, and takes a single drop of blood onto a piece of blotter paper. That minuscule sample provides enough material for a battery of tests, identifying inborn metabolic and genetic diseases so pediatricians can begin treating them immediately.

Unfortunately, most other clinical tests, and virtually all preclinical animal studies, require much larger volumes of blood or body fluids. Handling those larger volumes raises a host of logistical, technical, and ethical problems. Laboratory animals have little blood to give, so they can only provide a few samples before they die. Whole blood and serum must be shipped on dry ice, drastically inflating the costs of global clinical trials. Studying newborns or intensive-care patients with severely limited blood volumes has been difficult or impossible. Until now.

Aided by the increasing sensitivity of modern analytical techniques, researchers have developed new ways to perform complex assays on tiny volumes of blood and body fluids. The field has fed a boom in the use of dried blood spots (DBS) in both preclinical and clinical studies, with drug developers quickly discovering new advantages—and a few limitations—of this promising approach.

A little dab will do
In an industry driven by high-tech advances, DBS is refreshingly old-school. “From the bioanalytical chemist’s perspective there’s nothing new or fancy about dried blood spots. What you’re doing is reconstituting a blood spot to quantify an analyte of interest out of it,” says Steve Michael, PhD, global vice president and chief science officer of global bioanalytical services at Covance in Princeton, N.J.

click to enlarge A technician punches dried blood spot samples from a card. (Image: ABSciex)

At its core, DBS relies on absorbent cards, paper punches, and simple extraction procedures. After the initial extraction, researchers use established small-volume chromatography methods to separate the analytes of interest. “You’re coming out with a small amount of sample, and then using techniques [such as those] we’ve developed over time to handle this small sample and get as much information out of it as possible,” says Don Arnold, general manager of the Eksigent division of AB Sciex in Dublin, Calif.

For biopharmaceutical analyses, the key breakthrough has been further downstream, where drastic increases in the sensitivity and selectivity of mass spectrometry have enabled highly sophisticated analyses on vanishingly small DBS samples.

With the newfound ability to track tiny changes in these tiny samples, drug discovery and development teams are poised to reap numerous benefits. “The DBS analysis really started from a preclinical animal standpoint,” says Chris Evans, PhD, section manager of the bioanalytical sciences and development group at GlaxoSmithKline in King of Prussia, Pa. Evans adds that “the animal handlers were able to collect less blood from a rodent, where we’re limited to the amount of blood that can and should be collected at a time.”

Previously, investigators often had to sample and sacrifice one group of animals early in the experiment and save another group for sampling and sacrificing later. By taking single drops of blood instead of whole syringes, preclinical researchers can now track metabolic and pharmacodynamic changes in each animal over time. That allows them to reduce the number of animals in each experiment, and also provides better data.

While blood volume is seldom a major limitation in clinical trials, the invasiveness of phlebotomy certainly is. With DBS, “instead of having a cannula inserted in the forearm vein of the subject, we can have a finger or a heel prick, which is much less invasive. This allows easier recruitment of patients, especially seriously ill patients,” says Luca Ferrari, bioanalysis site head at Aptuit in Verona, Italy.

Besides making trials more patient-friendly, DBS allows minimally-trained staff to take samples, and the samples themselves can be shipped at ambient temperatures in an envelope. Those advantages can be especially important in large global trials and studies done in remote areas with poor medical coverage. According to Ferrari, DBS can also improve the quality of the data: “[DBS] does not require the need to centrifuge, subaliquot, freeze, and defrost the samples, all of which can introduce errors into the analysis.”

click to enlarge After the sample is extracted, researchers use established chromatography methods to separate the analytes of interest. (Image: Covance)

Sticking points
Despite its many advantages, even proponents concede that DBS has some distinct limitations. Because of the small sample volume, for example, some analytes might be out of reach. “If the target sensitivity is a limit even using conventional techniques like plasma or blood sampling, it could be a problem for DBS, because the amount of matrix that is available is even less. Nevertheless, the sensitivity loss is now less of a limitation due to improved analytical techniques,” says Ferrari.

Assays for DBS can also be more time-consuming and complex to develop, as the method requires the latest analytical technology. Results from these new assays might also be difficult to compare with earlier work that used traditional blood or plasma samples, so researchers may need to do bridging studies to determine how to compare data from new and old experiments.

Dried blood spots are also blazing a new trail with regulatory agencies, none of which have issued definitive rulings on how they’ll handle new drug applications that use the technique. While liquid assays are covered by a long list of validation standards and extensive international guidance, some of those standards clearly do not apply to dried blood spots. For example, a standard step in validating traditional liquid assays is testing their reproducibility after freezing and thawing the sample. That test is meaningless for DBS, where the sample is dried and stored at room temperature.

Another problem stems from the nature of blood, and how it behaves when dried. The size of a blood spot depends partly on the patient’s hematocrit, the percentage of the blood that is made up of red blood cells. Hematocrit varies from person to person, and even in one person from day to day. Each sample is therefore diluted across the paper to a different degree. Technicians sampling the same size piece of paper from each blood spot may see wildly different analyte levels even if the actual circulating analyte levels are the same.

The high sensitivity of modern assays also mandates more stringent standards for the blotter paper. “Dried blood spots for drug development are quantitatively much more demanding, so for these cards we’re monitoring and controlling the properties of the card ... down at the three millimeter scale, which is the size of the disc that many people are using,” says James Robbins, PhD, field application scientist at the Whatman division of GE Healthcare in Piscataway, N.J.

Wiping away tears
Blood isn’t the only body fluid amenable to spotting on cards. “It’s a technique that you can spot or analyze other limited-volume fluids, such as synovial fluid, tears, and [cerebrospinal fluid],” says Shane Needham, PhD, laboratory director at Alturas Analytics in Moscow, Idaho.

click to enlarge After the sample is extracted, researchers use established chromatography methods to separate the analytes of interest. (Image: Covance)

As a contract research organization, Alturas has developed several new techniques to solve the unique problems associated with these other fluids. One major issue is that colorless liquids leave colorless spots, which makes it harder for technicians to sample the correct portion of the card. Alturas addresses that with proprietary paper treatments that show the spot locations with a color change. Whatman also offers color-changing papers for these types of analyses.

Taking human technicians out of the process could also help minimize variability, especially when the spots are difficult to locate. “Currently the punching of the cards in order to do the extraction is primarily manual,” says GE’s Robbins. He adds that GE, in collaboration with Hamilton, is developing an automated system for punching discs and extracting analytes. Several other companies are developing a variety of disc-punching automation approaches.

Despite the challenges, clear body fluids may avoid some of the problems that can trip up dried blood spot analysis. “There’s certainly not the variability that you would have with blood, because there’s no hematocrit [variation],” says Needham.
Indeed, the new method may even let researchers do experiments that were previously impossible. “There have been times in the past where researchers have wanted to collect these fluids ... and they didn’t because there wasn’t a good technique for collection, storage, and sampling,” says Needham, adding that “we’ve actually seen some programs be resurrected because they could sample the proper compartment in the in vivo system.”

As an example, he points to arthritis, which is most likely to affect biomarkers in synovial fluid. Unfortunately, rodent preclinical models only have a few microliters of synovial fluid in each joint, forcing drug developers to rely on surrogate markers in the animals’ plasma. With dried matrix spotting, those arthritis drug campaigns can now sample the joints directly.

While experts throughout the field concede that dried body fluid spotting still faces serious challenges, they are generally optimistic about the technique. “Just like 25 years ago the FDA was hesitant to accept LC-MS/MS analyses, but researchers proved time and time again that it is a verifiable, validatable technique that is accurate and precise ... that’s what’s going to happen with dried blood spots. It’s going to be part of bioanalysis in the future,” says Needham.

About the Author
Originally trained as a microbiologist, Alan Dove has been writing about science and its interfaces with industry and government for more than a decade.