Dried Blood Spot

In 1913, for the first time, Ivar Bang described a dried blood matrix as an alternative sampling method. This alternative sampling method, based on collecting blood spots on cellulose paper and drying them, is called “dried blood spot” or DBS testing ⁹. Later in 1963, Robert Guthrie used this technique for neonatal screening to detect inborn errors of metabolism, phenylketonuria ¹⁰. Since then, the use and analysis of DBSs has been further applied towards more than thirty disorders and saved many lives in both developed and developing countries ¹¹.

Compared to other bio-fluids, the DBS sampling technique is minimally invasive, requires small blood volumes (approx. <50 µL), is inexpensive and has a higher analyte stability. Moreover, the possibility of self-sampling at home with minimal training, and

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transportation through mail, allows its utilization even in resource-poor settings. The DBS sampling technique has been widely applied for neonatal screening, epidemiological disease surveillance, and is also gaining attention in preclinical drug development, toxicokinetic and pharmacokinetic (PK) studies, clinical pharmacology, targeted and nontargeted metabolic profiling, therapeutic drug monitoring (TDM), and forensic toxicology¹¹. With the inherent advantages of the DBS sampling technique compared to the plasma based strategies, DBS based assays may play a critical role in precision medicine for the analysis of biomarkers or pharmaceuticals for disease prevention and treatment.

1.2.1 DBS analysis methods

For decades, chromatography and mass spectrometry have been utilized as the major tools in DBS analysis for pharmaceutical and metabolomics analysis ¹¹. In 1976, the first application of mass spectrometry (MS) to DBS analysis was reported for fatty acid determination by direct chemical ionization ¹². Later in the mid-1980’s, gas chromatography (GC) was the technique of choice for the separation and analysis of volatile small molecules, and derivatized fatty acids were measured from DBS samples using GC-MS¹³. After the electrospray ionization (ESI) method became commercially available, DBS based newborn screening laboratories began to use liquid chromatography-tandem mass spectrometry (LC-MS/MS) techniques. Currently, about 121 distinct biomarkers were determined from DBS samples utilizing MS technology encompassing both LC-MS/MS and GC-MS/MS ¹⁴.

However, due to its limited applicability for gas soluble, volatile and heat resistance small molecules (often derivatization is required to turn non-volatile molecules to volatile), GC was only utilized moderately compared to LC. Moreover, the improved

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specificity and sensitivity with a significantly faster analysis afforded by LC-MS/MS methods offers additional benefits for the analysis of blood spot analysis compared to GC-MS¹⁴. DBS analysis incorporated with two dimensional chromatography (2D-LC-MS/MS) and on-line sample extraction improves analytical sensitivity, quantitation accuracy, and reduces matrix and carry-over effects ^15-16. The sensitivity of DBS assays for both polar and non-polar analytes were also enhanced by the advances in ion source technology ^17-18. The compound specific transitions used for both selective/multiple reaction monitoring (SRM/MRM) modes in MS/MS detection have advanced the DBS assay specificity remarkably along with improving linearity and limits of detection¹⁹.

1.2.2 DBS clinical applications

The DBS sampling technique has been widely applied for large scale neonatal screening and microbiological and epidemiological disease surveillance. Recently it is gaining attention in preclinical drug development, PK, and TDM, clinical pharmacology, targeted and non-targeted metabolic profiling, forensic toxicology, and doping or environmental contaminant control

The use of DBS in preclinical studies results in a reduction in the volume of blood collected, which has a significant impact on animal studies. Using the DBS sampling technique, the number of rodents needed for each study can be reduced by up to 75%.

Moreover, DBS sampling in preclinical studies contributes to high quality data because more time points can be added without the need for additional rodents. Therefore, the DBS sampling technique is highly suitable for the collection of samples during preclinical PK and TK studies. DBS sampling agrees with the principles of the 3R’s (replacement, reduction and refinement), as fewer animals are needed ^20-21.

Application of DBS sampling is an emerging technique for TDM. TDM can be

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performed either by monitoring the drug, the biomarker, or their metabolites or, monitoring the viral load after administering the drugs for viral infections directly on the DBS ¹¹. Monitoring drug efficacy ensures early detection of emerging drug resistance (for example in HIV), and can promote a transition to a different drug ¹¹. TDM assays for drugs are usually performed in serum or plasma, obtained by venous blood sampling. Due to its numerous advantages, DBS sampling could be a favorable alternative. Nevertheless, the interpretation of drug concentration measurements for TDM are usually based on reference ranges established in plasma or serum samples. Therefore, there is a need to convert the information obtained from DBS to plasma levels. The DBS sampling technique has been applied for the analysis of a wide range of therapeutic drugs for TDM including: ACE inhibitors, analgesics, antibiotics, antiepileptic drugs, antidepressants, anthelmintic drugs, antimalarial drugs, antifungal drugs, antiretroviral drugs, diuretic drugs, histamine H2-receptor antagonists, immunosuppressant drugs, chemotherapeutic drugs, statin, β-blockers and μ-opioid agonists ²².

For forensic toxicology analysis, whole blood is an important specimen, because it provides information on the type of substances and the amount of substances in users at the time of collection ²³. Since DBS is a dried form of whole blood and only requires a few micro liters for analysis, using this sampling technique for forensic toxicology may be beneficial in cases involving highly decomposed bodies, or in which fluids are minimal.

Additionally DBS is a quick sampling technique which is especially important in cases which analyze samples containing analytes with short lifetimes, such as heroin and its metabolite, 6-acetylmorphine (6-AM) ²⁴.

DBS-based metabolomics is an emerging technique towards precision medicine.

The developments in this rapidly developing field may bring about many contemporary discoveries, among which are metabolite-biomarkers for diseases. DBS represents an

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alternative as it overcomes many of the current plasma metabolome issues such as the stability of metabolites in plasma or blood samples after acquisition, invasive sampling collection, and the sophisticated cold chain storage of bio-fluids. Moreover, bio-fluid spotting has been shown to be a potential alternative to conventional plasma in metabolite profiling using UPLC-MS and GC-MS ^25-26. Yet, to establish the DBS sampling as a powerful tool for clinical metabolomics applications, a lot more research has to be done to make DBS sample analysis compatible with the gold standard plasma sample analysis.

1.2.3 Challenges associated with DBS sampling technique for clinical applications Despite its simplicity and numerous advantages, the real world application of the DBS sampling technique still faces many critical challenges, including inadequate sampling and contaminated samples with unacceptable quality by self-sampling, lack of sensitivity due to the small sample volumes, variation in the amount of spotted blood volume, the hematocrit (Hct) variation effect and requirement of additional clinical validation to establish DBS methods for clinical practice in replacement of the gold standard plasma sampling ^{20, 22}.

Minimal training in blood spotting and handling of spotted samples can resolve inadequate sampling problems and issues with contamination during sampling. The sensitivity of DBS assays could be improved by developing analytical methods based on MS, either using GC-MS or LC-MS/MS, which is associated with high sensitivity and specificity¹². In general, it is difficult to control the spot volume for DBS sampling, unless special sampling equipment is applied, however the application of special equipment for DBS sampling conflicts with its major advantage patient self-sampling and cost effectiveness, and may not be feasible for clinical practice. Although the spot volume variation problem could be resolved by using a fixed diameter subsample, one potential

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problem is the Hct variation caused bias. Hct is the volume percentage of red blood cells (RBC) in whole blood and it shows large individual variability between different genders, age, physical status and nutritional conditions of the subjects. Predominantly, Hct affects the viscosity of blood by causing differences in spot formation, spot size, drying time, and homogeneity, which leads to analytical uncertainty in terms of precision, recovery and accuracy; it also further influences the quantification accuracy of clinical samples as the blood to plasma ratio of the drugs varies ²⁰. Therefore, to resolve the effects of spot volume and Hct variation on spot formation, whole spot analysis should be applied.

Recently, Liao et al., developed a post column infused-internal standard (PCI-IS) method to estimate and calibrate the blood volume on DBS cards ²⁷. The developed method estimated the blood volume by measuring the total salts in blood samples using the PCI-IS. The developed method is able to facilitate whole spot analysis.

In clinical practice, plasma sampling has been the gold standard for many decades and many databases on drug monitoring and biomarker identification and quantification were established using data from plasma samples. Therefore, investigating the concentration relationships between DBS and plasma samples is important when DBS samples are utilized for the replacement of plasma measurements. Since the main difference between these two sampling techniques are the composition of sample, direct correlation of DBS and plasma concentrations could display large deviations. Two main factors that cause the large deviation in correlating DBS and plasma concentrations are Hct variation and plasma to whole blood drug distribution ratio. Emmons and Rowland proposed an equation to bridge the concentration gap between plasma and DBS samples on the basis of blood-to-plasma drug concentration ratio, Hct value and unbound fraction in plasma ²⁸. Recently, several bridging studies were developed for anti-HIV, anti-cancer, antiepileptic and antidepressant drugs to establish the concentration relationships between

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DBS samples and plasma samples ^29-32. Therefore, it is necessary to perform clinical validation when DBS samples are being used instead of plasma samples.