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.
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.
In the last five years, investigations of the gut microbiome, or microbiota, have skyrocketed largely due to advances in massively parallel sequencing technology and bioinformatic approaches such as the reconstruction of transcriptomes using de novo assembly in the absence of complete reference genomes. These metatranscriptomic studies of stool samples have identified and cataloged bacteria, fungi, and viruses that are common to healthy colonic microbial populations. For instance, over 35,000 bacterial species comprise the gut microbiota; although organisms belonging to the phyla Firmicutes and Bacteroidetes predominate1. Microbiome researchers have poured in a substantial body of work associating the dysbiosis of the microbiome with many pathologies including metabolic diseases, colorectal cancer, multiple sclerosis, cognitive developmental disorders, and autoimmune diseases2-10.
There has been a wave of companies rushing to leverage these connections to develop novel therapeutics. In fact, over 20 well-funded start-ups have recently surfaced with the mission to harness the power of the microbiome to treat and prevent disease. Of these microorganisms-orientated companies, Vendanta Biosciences (http://www.vedantabio.com/) is tackling solid tumors with bacteria, Seres Therapeutics (http://www.serestherapeutics.com/) is optimizing fecal transplants to treat recurrent Clostridium difficile infections, and Blue Turtle Bio (http://blueturtlebio.com/) is also utilizing bacteria from the gut microbiome but as a drug delivery platform. A Swedish company, Infant Bacterial Therapeutics (http://ibtherapeutics.com/) (IBT), which is taking on necrotizing enterocolitis (NEC) with Lactobacillus reuteri-based candidates, has been granted the Rare Pediatric Disease Designation by the FDA for its lead drug candidate to prevent NEC – a disease, which is fatal, especially for premature neonates.
The modulation of the gut microbiota composition of infants and children has been investigated as a therapeutic route for atopic dermatitis, bacterial gastroenteritis, inflammatory bowel disease, necrotizing enterocolitis, and allergic diseases11. Probiotic interventions encourage the growth of commensal bacteria and ward off the colonization of pathogenic organisms, thereby affording protection to the intestinal barrier function and reducing food allergies and atopic disease incident rates12. Infant formula containing low dose Bifidobacterium lactis supplementation is shown to provide similar early life outcomes to breastfeeding with regards to gastrointestinal infection rates and immune system and gut maturation13. However, benefits of probiotic usage are not limited to infants.
The Probiotics in Pregnancy Study conducted in Wellington and Auckland, New Zealand involved the administration of Lactobacillus rhamnosus four hundred pregnant women during pregnancy and breastfeeding. This study showed reduced rates of infant eczema and atopic sensitization at 12 months but also decreased rates of material gestational diabetes mellitus and the presence of bacterial vaginosis and vaginal carriage of Group B Streptococcus, and postpartum depression and anxiety14.
The potential of leveraging the data that can be gathered through deep microbiome profiling is strong and is increasingly becoming a promising strategic approach for drug discovery and development companies to treat infant, pediatric, and adult diseases.
- Jandhyala, S. M., Talukdar, R., Subramanyam, C., Vuyyuru, H., Sasikala, M., & Reddy, D. N. (2015). Role of the normal gut microbiota. World Journal of Gastroenterology: WJG, 21(29), 8787. (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4528021/pdf/WJG-21-8787.pdf)
- Armougom, F., Henry, M., Vialettes, B., Raccah, D., & Raoult, D. (2009). Monitoring bacterial community of human gut microbiota reveals an increase in Lactobacillus in obese patients and Methanogens in anorexic patients. PloS one, 4(9), e7125. (http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0007125)
- Brown, C. T., Davis-Richardson, A. G., Giongo, A., Gano, K. A., Crabb, D. B., Mukherjee, N., & Hyöty, H. (2011). Gut microbiome metagenomics analysis suggests a functional model for the development of autoimmunity for type 1 diabetes. PloS one, 6(10), e25792. (http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0025792)
- Larsen, N., Vogensen, F. K., van den Berg, F. W., Nielsen, D. S., Andreasen, A. S., Pedersen, B. K., & Jakobsen, M. (2010). Gut microbiota in human adults with type 2 diabetes differs from non-diabetic adults. PloS one, 5(2), e9085. (http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0009085)
- Weir, T. L., Manter, D. K., Sheflin, A. M., Barnett, B. A., Heuberger, A. L., & Ryan, E. P. (2013). Stool microbiome and metabolome differences between colorectal cancer patients and healthy adults. PloS one, 8(8), e70803. (http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0070803)
- Zhang, Y. J., Li, S., Gan, R. Y., Zhou, T., Xu, D. P., & Li, H. B. (2015). Impacts of gut bacteria on human health and diseases. International journal of molecular sciences, 16(4), 7493-7519. (http://www.mdpi.com/1422-0067/16/4/7493)
- Jangi, S., Gandhi, R., Cox, L. M., Li, N., Von Glehn, F., Yan, R., & Cook, S. (2016). Alterations of the human gut microbiome in multiple sclerosis. Nature Communications, 7. (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4931233/)
- Cao, X., Lin, P., Jiang, P., & Li, C. (2013). Characteristics of the gastrointestinal microbiome in children with autism spectrum disorder: a systematic review. Shanghai Arch Psychiatry, 25(6), 342-53. (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4054584/)
- Lyte, M. (2013). Microbial endocrinology in the microbiome-gut-brain axis: how bacterial production and utilization of neurochemicals influence behavior. PLoS Pathog, 9(11), e1003726. (http://journals.plos.org/plospathogens/article?id=10.1371/journal.ppat.1003726)
- Brusca, S. B., Abramson, S. B., & Scher, J. U. (2014). Microbiome and mucosal inflammation as extra-articular triggers for rheumatoid arthritis and autoimmunity. Current opinion in rheumatology, 26(1), 101. (https://www.ncbi.nlm.nih.gov/pubmed/24247114)
- Awasthi, S., Wilken, R., German, J. B., Mills, D. A., Lebrilla, C. B., Kim, K., & Maverakis, E. (2016). Dietary supplementation with Bifidobacterium longum subsp. infantis (B. infantis) in healthy breastfed infants: study protocol for a randomised controlled trial. Trials, 17(1), 340. (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4957407/)
- Cao, S., Feehley, T. J., & Nagler, C. R. (2014). The role of commensal bacteria in the regulation of sensitization to food allergens. FEBS letters,588(22), 4258-4266. (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4216641/)
- Baglatzi, L., Gavrili, S., Stamouli, K., Zachaki, S., Favre, L., Pecquet, S., & Costalos, C. (2016). Effect of infant formula containing a low dose of the probiotic Bifidobacterium lactis CNCM I-3446 on immune and gut functions in C-section delivered babies: a pilot study. Clinical medicine insights. Pediatrics, 10, 11. (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4792197/)
- Barthow, C., Wickens, K., Stanley, T., Mitchell, E. A., Maude, R., Abels, P., & Hood, F. (2016). The Probiotics in Pregnancy Study (PiP Study):rationale and design of a double-blind randomised controlled trial to improve maternal health during pregnancy and prevent infant eczema and allergy. BMC pregnancy and childbirth, 16(1), 133. (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4891898/)
Editor’s Note: The original article first appeared on the Ocean Ridge Biosciences website, which, after the acquisition of Ocean Ridge Biosciences, was modified for use on the Frontage website.