Frontage Labs

Microsampling and Dried Blood Spot (DBS) Techniques in Bioanalytical Assays

Microsampling Overview 

Dried blood spots (DBS) and capillaries/capillary microsampling are the two most used microsampling techniques. In this article, we will give an overview of current microsampling techniques, the advantages, and challenges involved, and showcase how microsampling can generate high-quality bioanalytical data and accurate TK/PK profiling. Microsampling is a process through which low-volume samples (<100 μL) of fluid from the human body are captured for analysis. This enables minimally invasive analyses of samples when they are present in low volumes. 

First used by Dr. Robert Guthrie for detecting metabolomic disorders in newborns in the 1960s, the dried blood spot (DBS) technology allowed routine neonatal testing for phenylketonuria (PKU) worldwide. In the drug discovery world, DBS was not considered for pharmacokinetic/toxicokinetic (PK/TK) studies until recently. In a 2010 study, a 10-time-points serial mouse PK study was done using microsamples and DBS, showing that serial microsampling and DBS provided quality serial PK profiling data in comparison with plasma-based composite studies. It was determined over time that DBS can reduce the sample volume needed by 75% and generate reliable PK data for use. 

Microsampling in Bioanalysis 

Current pharmacokinetic and toxicological studies have an increasing demand for the volume of blood required from study animals, especially when the study involves small rodents. A typical toxicology study has satellite groups of rodents, with each group having multiple animals, to achieve enough volume for analysis. Individual variance between animals can be high and animal physiology is affected at each draw. Altogether these affect the accuracy and efficiency of the studies. 

The FDA ICH M3 guidance recommends a reduction of “[…] the use of animals in accordance with the 3R (reduce/refine/replace) principle” and microsampling techniques help preclinical researchers follow this. As a response, microsampling techniques have been introduced into bioanalytical studies. In a poll conducted during our webinar on DBS and Capillary Microsampling, a majority of the respondents reported ethical concerns and efficiency to be their drivers of interest in microsampling for bioanalysis. 

Advantages of Microsampling 

Previous studies have shown that microsampling greatly reduces blood sample volume needed by 80%. Because of this, fewer rodents are needed when conducting toxicokinetic (TK) studies, a number reduced by 75%. This reduction has allowed for serial TK/PK profiling, whereas when conventionally used plasma samples were collected, only composite profiling had been possible. During composite profiling, several animals need to be bled at different time points, and the large variability between animals added another factor to consider. Serial profiling also allows the generation of much more reliable and consistent PK profiles. For clinical studies, DBS sampling is especially patient-friendly for pediatric or studies with critically ill patients. 

Handling DBS samples is also much simpler than traditional procedures. There is no centrifugation needed as is required when harvesting plasma and there are no requirements to aliquot or freeze samples as with plasma, saving freezer space and eliminating the need for cold storage during shipment. DBS sampling is also a safe technique because all pathogens, including HIV and HepB, are inactivated upon contact with the filter paper used. This ensures as well that the personnel handling the DBS samples are also safe. The costs of such methods are not low simply because of a reduction in R&D costs due to low volumes and fewer rodents needed. In fact, because DBS samples are not considered biohazardous and do not require special carriers, shipping costs are lower than they would be for plasma samples. 

Challenges of Microsampling

In a poll conducted during our webinar on DBS and Capillary Microsampling, greater than 60% of the attendees reported “sample size” as their biggest challenge during method development for microsample bioanalysis. While assaying with smaller sample volumes is desirable for many reasons, the low volume limits sensitivity testing. 

60% of the attendees reported “sample size” as their biggest challenge during method development for microsample bioanalysis.

Frontage Webinar Respondents, DBS and Capillary Microsampling

Differences in the sample quality can also be seen in spot-to-spot variation. The hematocrit (Ht) effect refers to the volume of red blood cells in the blood sample which can affect how well the blood spreads on the filter, causing the spot size to be either bigger or smaller. This can affect the quality and reliability of the blood samples as well as the quantification of the sample. 

While most compounds are stable in DBS, compound stability can be an issue when handling compounds that are unusual or unstable. In such cases, applying inhibitors or stabilizers is inconvenient due to the design. 

Choose a Partner with Microsampling Experience

When assessing DBS for LC-MS/MS method validation, it is critical not only to validate the parameters traditionally validated for plasma samples but also to additionally evaluate the spot size, sampling location, and hematocrit effect. DBS is a special kind of matrix because, unlike other matrices, it cannot be simply vortexed or diluted and homogeneity is difficult to ensure. At Frontage, we have developed special preparation and extraction techniques to prepare standards and QCs to ensure robust performance and produce clean data when working with microsampling techniques. We have developed a unique sample-handling method utilizing pre-scored capillaries developed in-house, at Frontage Laboratories (PA, USA). Learn more by reading our case study on this technique, and how it overcomes some of the limitations of the conventional capillary microsampling approach and supports regulated bioanalytical studies. 

Case Study: Capillary Microsampling (CMS) Technique for Low-Volume Bioanalytical Plasma Analysis in Support of a Regulated Study: Frontage bioanalytical scientists developed a novel procedure for the collection and isolation of microvolumes of plasma using plastic instead of glass capability tubes to overcome issues associated with the typical glass CMS technique. 

What are DART studies? 

DART, or Developmental and Reproductive Toxicology, studies assess critical information about drug exposure effects before and during mating, and evaluate maternal and fetal changes during gestation, identifying any alterations in the development of the progeny following birth. In essence, these studies elucidate the toxicity effects of the compound in question.

Why are the challenges of assessing risks by toxicity? 

Depending on when the exposure takes place, different organs or aspects of development may be affected and these periods of time are the critical periods of susceptibility. Toxicants can be made up of various agents initiating a wide variety of mechanisms in the body. They may also affect only certain populations, based on genetic differences or varying health histories. The potential mechanism of action of the toxicant must be understood at many levels of the biological organization (pathways, cells, etc.) to truly understand the overall processes of developmental toxicity. 

How are DART studies different from general toxicology studies? 

DART studies are not separate from different from standard toxicology in that they have the same application of toxicological principles such as toxicokinetics (TK), pharmacokinetics (PK), ADME, and other such parameters. There is however special emphasis on the timing and duration of exposure, dose level, and the consequence on the mother and fetus both. 

Why are DART studies essential? 

DART studies assess information about how exposure to a drug effect different developmental stages. Such altered development may be manifested as death, malformation, growth retardation, or functional deficits. FDA guidance recommends certain international standards for the harmonization of the assessment of nonclinical developmental and reproductive toxicity testing and deems that these studies are required before human clinical trials and marketing authorization. In order to initiate clinical trials for women of child-bearing potential (WOCBP), the FDA requires the initiation of preclinical developmental and reproductive toxicology (DART) studies using mammalian research models.  

What is the typical DART program? 

The typical species for DART studies include rats and rabbits via multiple routes of administration but other research models are also available for DART, such as mouse, hamster, dog, minipig, and monkey. There are a few types of DART studies and each focus on studying a different phase of the developmental process: 

  • Fertility and Early Embryonic Development (FEED) study (Segment I) evaluates the effects on mating, estrous cyclicity, spermatogenesis, and ability to produce a viable implant. At Frontage, we assess sperm count, motility, and morphology are assessed using a Hamilton-Thorne Analyzer. 
  • Embryofetal Development (EFD) study (Segment II) demonstrates the effects of gestational exposure on the pregnant female and the developing fetus during the organogenesis period. This study evaluates fetal development and survival. 
  • Pre- and Post-natal (PPND) study (Segment III) quantifies the effect of gestational and lactational maternal exposure on embryonic development, the process of parturition, and the development of the offspring. 

For more information on the dosing parameters, definitive study details, and study timelines, watch our webinar: Guidelines for Developmental and Reproductive Toxicity (DART).

When should we conduct DART studies? 

The EFD and FEED designs can be run in parallel and are generally required for Phase 2 Clinical Trials. PPND studies are usually required for Phase 3 Clinical Trials but can be done earlier if needed. 

A Partner for DART Studies 

Choosing a CRO like Frontage offers you access to highly trained expert scientists with a long history of successfully performing specialized toxicology studies, including regulated DART studies. At Frontage, we offer single and multi-generation studies and have the capability to conduct DART studies following the ICH S5 (R3) Guideline. Request a quote for a DART study


  • Developmental and Reproductive Toxicology: A Practical Approach (Third Edition) by Ronald Hood