Dried Blood Spot analysis in drug discovery and development

Developments in biomedical sciences have revolutionized understanding of human physiology and underlying mechanisms of ailments. The 20th century has witnessed extraordinary benefits of medicines which have significantly improved quality of life for millions of people across the globe. Nevertheless, there remain a massive unmet medical need which prompts the scientific community to constantly work towards discovery and development of new ‘magic bullets’.

Drug discovery and development demands greater resources and time so as to deliver efficient and safe drugs. With the increasing cost and time lines for drug discovery and development, there is need for innovation at every stage of this cycle. Pre-clinical stage of drug discovery involves studies such as pharmacodynamics (PD), efficacy, pharmacokinetics (PK) and toxicokinetics (TK) in various animal species while clinical development involves in-vivo testing and assessment of similar parameters in humans.

Across all development stages, most of the time, ‘blood’ is always a centre to measure ‘what body does to drug’ and ‘what drug does to body’. Various regulatory agencies such as US Food and Drug Administration (USFDA) have also endorsed ‘whole blood’ as appropriate and acceptable bio-matrix to evaluate and monitor these activities for regulatory filings . However, due to ease of handling, plasma has been used as ‘gold standard’ to assess and correlate both PD and PK of the drug in pre-clinical and clinical development. Technically, DBS methodology involves qualitative and / or quantitative measurement of drug / new chemical entity of interest, corresponding metabolites, various physiological / pharmacological / disease related diagnostic, genomic, efficacy and safety molecular biomarkers.

Bioanalysis is a central function that addresses these issues and faces numerous scientific and technological challenges. Traditional ‘plasma analysis’ bioanalytical strategy demands larger blood samples to be withdrawn from study subjects (animals / human), and thus directly affects pre-clinical and clinical study designs. For example, to conduct PK / TK studies in pre-clinical animal species (mouse and rat), the need for appropriate volume of plasma to be used in bioanalysis leads to a composite study design involving parallel sampling regimen . This essentially increase number of animals required for the study and may also lead to variability in observed data as different animals are used for different time points. In case of clinical studies, development of new drugs for paediatrics is not fully addressed due to ethical issues in obtaining large blood samples from this ‘special population’.

Dried Blood Spot  technology
Collection of very low volume blood samples on absorbent paper, known as DBS technique, was first reported by Guthrie and Susi in 1963 . The technique involved collection of drop of blood via a heel prick and was first used to measure phenylalanine in newborns for the detection of inborn errors of metabolism (phenylketonuria).

Although the technique was reported around forty years ago, its application in qualitative / quantitative bioanalysis is of recent research interest. In last decade, there has been a logarithmic development in the bioanalytical technologies that have made highly sensitive and selective analytical instruments for bioanalysts.  Evolution in mass spectrometry and ultra-high pressure / nano-liter scale liquid chromatography systems have enabled scientist to handle very low volume of biomatrix for bioanalysis and yet achieve highest sensitivity and selectivity.

Compared to conventional plasma based approach, DBS technology offers several advantages such as minimally invasive, enables micro-sampling, requirement of very small sample volume, minimal sample processing time (no centrifugation step), improved analyte stability, room temperature storage, no special transportation requirement and reduced biohazard risk.

DBS process essentially involves spotting of pre-measured volume of blood on to appropriate sampling paper. The scientific literature reported spotting volume in the range if 5-100 µL. However, in recent time the spotting volume has been in the range of 5-30 µL. The blood spreads on to paper and forms a circle and is allowed to dry. Sample pre-treatment and drying conditions depend on chemical nature of analyte and stability concerns (light vs. dark, controlled humidity, etc).

Spotted cards are dried for minimum of 3 hours and are packed in resealable bags containing desiccant to avoid unwanted exposure to moisture. Dried cards are stored at bench top in appropriate storage boxes (usually air tight containers). Storage of sampling cards is one of the most pronounced advantages of DBS technology as it enables room temperature storage. Analyte being in dry solid state, generally, is less prone to degradation. When compared to plasma storage, which requires refrigerated conditions for storage, DBS offers major advantage in terms of cost and ease as it negates the need for

special storage conditions. However, one can always assess storage stability of analyte on DBS cards if temperature related stability concerns are known for an intended analyte.

For sample processing and analysis, researchers have explored options of processing whole or part of spot (disc of specific diameter punched from DBS sample). The decision to use whole or part of spot depends on two issues, first the total volume of blood that is required to achieve desired sensitivity (Limit of quantification, LOQ), second, most recently and significantly to address the issue of variability induced by the hematocrit of the blood sample. It is logical to say that, varying hematocrit (e.g. 30 to 75 per cent) posses varying blood viscosity (due to different proportion of the red blood cells) leading to different spot diameter for a constant spotting volume (e.g. for 25 µL spotting volume, the blood with 30 hematocrit may give different spot diameter than blood with 70 per cent hematocrit).

DBS being a dried solid matrix, sample preparation in DBS technology is unique as compared to other conventional liquid matrices (plasma / serum / urine / liquid blood) used in the bioanalysis (kind of solid – liquid extraction). The process involves addition of suitable solvent (based on logP and solubility of analyte of interest), usually HPLC grade water, methanol or acetonitrile. In contrary to conventional bioanalytical methods, the internal standard (IS) addition in DBS analysis is external i.e. added to the extraction solvent. The methodology is still in debate whether to add IS to blood before being spotted which will track overall extraction variability as compared to external addition which only track the instrument performance and its variability.

Further, DBS spots along with extraction solvent (containing IS) is vortexed or sonicated for analyte extraction for appropriate time. The extraction time is optimized for recovery of the analyte. After extraction, most of the time, the extraction tubes are centrifuged and supernatant is directly injected in to analytical instrument coupled with sensitive detector. However, depending on the assay requirement, towards fulfilling the requirements of the selectivity, sensitivity and ‘matrix effect’ related issues, the secondary sample treatment such as Solid Phase Extraction is employed (to aid analyte enrichment and / or getting rid of unwanted matrix interferences). An account of literature indicates use of analytical instruments such as HPLC coupled with UV, fluorescence, PDA detectors; however, use of liquid chromatography coupled with mass spectrometry is the most preferred analytical technique for the DBS analysis.

As far as  DBS method validation is concerned, there is no formal regulatory guidance available at the moment. However, conventional validation parameters such as accuracy, precision, linearity, selectivity, sensitivity, robustness and stability are been reported for many of the DBS assays. Along with these parameters, most recently, the emphasis has been given to assess additional parameters such as appropriateness of suitable sampling paper (treated Vs untreated cards), spotting volume and punch size, effect of hematocrit, evaluation of blood to plasma ratio, over the curve dilution methodology (dilution integrity for concentrations above the upper limit of quantification, ULOQ) etc.

DBS in pre-clinical drug discovery
Pre-clinical development involves use of experimental animals. Worldwide, there has been a ‘3R’ drive, implementing ‘Replacement, Refinement, Reduction’ principles in experiments involving animals [9]. It suggests where ever possible identify avenues for replacement for animal use, refine experimental protocols to avoid stress and pain to animals and finally, achieve reduction in animal usage. DBS methodology has potential to practically address these principles. The DBS technology implies micro sampling thus very well supports ‘serial blood sampling’. This essentially means obtaining all the study time points from a single animal. The refined approach of serial sampling coupled with DBS technology could significantly reduce the number of animals required for a given study .

In pre-clinical development, it is imperative to conduct TK studies to assess further developmental potential of the lead molecules. Conventionally, samples for TK studies are collected from satellite group of animals kept along with main toxicology study animals. With DBS technology, the need for satellite animals can be completely removed as small blood samples at various time points can be easily collected without affecting animal health and potential toxicology observations. This is a very significant advantage of DBS methodology and helps scientists to comply with ‘3R’ guidelines and help reduce cost through animal and test compound requirements .

DBS in clinical drug development
DBS offers unique advantages in clinical drug development. One of the major areas where DBS can play significant role is paediatric drug development. Clinical studies in this ‘special population’ group face challenges from ethics committees to collect large amount of blood samples, especially from neonates . With very low requirement of blood volume, DBS technology enables paediatric investigations for all age groups. The population PK studies involving opportunistic blood samples employing DBS technology further aids in dose decision studies in this population.

DBS being a dry matrix and use of chemicals on sampling cards leads to inactivation of pathogens (e.g. bacteria, viruses) thus reduces the biohazard for the bioanalyst. This also allows room temperature shipment of blood samples. This is particularly advantageous for conducting multi-centred clinical trial studies. Further, if the patients are adequately trained, it can imply self collection of sample and patients can send their samples to the laboratory via postal services. This advantage of DBS is useful to conduct clinical studies in remote locations and requires minimal clinical supervision. Application of this technology is also useful in conducting the ‘Therapeutic Drug Monitoring’ studies, which avoids patient’s visit to laboratory, reducing their economic burden. Scientific literature is populated with many of these studies involving use of DBS technology for specific drugs in various clinical studies.

Future prospects
DBS technology has received considerable attention in recent past and understanding around its intricacies is currently being explored. No doubt it offers range of advantages over conventional plasma based methods; however, it is yet to receive a green signal from regulatory agencies to adopt in all stages of drug development. Increasing evidence and development of more scientific rational for DBS will help strengthen this technology for wider acceptance.