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Approaches and technologies offering potential solutions to the problems associated with DBS analyses
Neil Spooner Ph.D., C.Chem, FRSC
Founder & DirectorSpooner Bioanalytical Solutions Ltd
Senior Visiting Research FellowSchool of Life and Medical Sciences, University of Hertfordshire
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Effects of Blood Hematocrit Has an effect on the diameter of derived DBS sample, resulting in unknown volume in sub-punch of sample
May have an effect on the recovery of analyte
Result is reduced (unknown) analytical accuracy
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70%45%20%
Alternative Approaches for Collecting Dried Blood SamplesVariety of technologies aimed at collecting fixed volume of blood regardless of hematocrit, resulting in improved accuracy of determined concentrations
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Volumetric Absorptive Microsampling - Mitra
Hydrophilic porous material
Each Tip has a fixed, highly reproducible internal porous volume - 10 µL
◦ Regardless of blood hematocrit
Rapid wicking◦ Under 6 seconds)
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Simple workflow
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Simple to use
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Mitra volumetric sampling performance
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Denniff & Spooner (2014) Anal. Chem. 86, 8489-8495, Denniff et al (2015) J. Pharm. Biomed. Anal. 108, 61-69,
Spooner et al (2015) Bioanalysis 7(6), 653-659
• Human blood at different HCTs was spiked with 14C caffeine
• Tip oxidised to 14CO2
• Radioactivity determined by scintillation counting
hemaPEN™ Workflow
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For Research Purposes only - this technology has not been approved by the regulatory authorities
hemaPEN™ Construction
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For Research Purposes only - this technology has not been approved by the regulatory authorities
hemaPEN™ Volumetric Performance
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For Research Purposes only - this technology has not been approved by the regulatory authorities
Human blood at different
HCTs spiked with caffeine
(5mg/mL)
3 µL blood spotted onto 226
substrate using;
o Air displacement pipette
o Positive displacement
pipette
o Glass capillary
Aqueous extracts analysed by
HPLC UV/Vis (276 nm)
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Leuthold et al. (2015), Anal.
Chem. 87, 2068-2071
CapiTainer AB
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Activation
Channel filling
Inlet dissolving
Outlet dissolving
Outlet activation
Punch out
Lenk et al (2015)
Bioanalysis 7(16),
2085-2094.
…..and there’s more
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Approaches for Normalising Quantitative Data Obtained by Conventional DBS SamplingVariety of approaches aimed at correcting the measured concentration
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Add internal standard to DBS sample prior to extraction
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Abu-Rabie et al. (2015) Anal. Chem. 87, 4996-5003
Normalise data to hematocrit K+ determination in aqueous extract of 3-mm DBS punch using a routine clinical chemistry analyzer
•[K+] intracellularly >> [K+] plasma and > 99% of cells are RBCs
•[K+] are tightly controlled � no large interindividual variability
•K+ is universal
•K+ proved to be stable in DBS
•K+ easily measurable in DBS
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Capiau et al. (2013) Anal. Chem. 85, 404-410; De Kesel et al. (2014), Anal. Bioanal. Chem. 406, 6749
Raw Data
Corrected for HCT
Normalise data to hematocrit
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Capiau et al. (2016) Anal. Chem. 88, 6538-6546
Non-contact diffuse reflectance spectroscopy to
determine different hemoglobin forms
Normalise data to an endogenous component
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Determination of blood volume by conductance
Electrical conductivity measured by ring-disk electrode in 100 µL aqueous extracts of spotted blood
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Points to Consider Quality/accuracy of analytical data is dependant on the quality of the sample collected
◦ Blood hematocrit introduces a bias into quantitative data based upon lack of control of blood volume and recovery with conventional DBS sample collection approaches
Consider use of alternative blood collection approaches if;
◦ Determined data is to be compared between labs, or across time
◦ The differences between measured values for a positive and negative result are marginal
Can alternative technologies and workflows be introduced to further enable automation? Consider potential for;
◦ Increased data quality
◦ Cost savings
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