the use of blood microsampling for the quantitative
TRANSCRIPT
The use of Blood Microsampling for the Quantitative Bioanalysis of Pharmaceuticals in Clinical and Pre-clinical Studies
Neil SpoonerDrug Metabolism & Pharmacokinetics, Ware, UK
What is Blood Microsampling?
�Reduced volumes of blood collected from humans and animals for the determination of circulating blood concentrations of dosed drug substance, or other entities (biomarkers, etc)–<<100 µL compared to significantly >200 µL taken conventionally
What are the Benefits of Microsampling?
�Has ethical 3Rs (replacement, reduction, refinement) implications, particularly in rodents
–Reduced numbers of animals due more time-points being taken from a single animal
–Reduced requirement to warm rodents to facilitate collection of required blood volume
� Improved data quality in rodents – serial rather than composite profiles
�Facilitates pediatric & other clinical studies where availability of blood volumes may be limited
�Potential for simplified clinical sample collection – ‘finger prick’ approach–Improved recruitment, particularly where PK is the only sample
being taken - PopPK
What are Dried Blood Spot (DBS) Samples?
�Easy way of collecting, shipping & storing blood samples�Technique has been around for >40 years
–Widely used in new born screening, therapeutic drug monitoring & for trials in remote areas, e.g. anti-malarials
�Blood from animal / human spotted onto collection card�Cards air dried & stored / shipped desiccated at room temperature�Discs punched out of the DBS sample for quantitative analysis
–Sub-punch allows for the spotted volume not being accurate–Accuracy attained through the fixed punch diameter
Further Benefits of DBS Sampling
�Simplified processes
–Removes need for centrifugation or sub-aliquotting of plasma
–Shipping & storage at ambient temperature means that dry ice & freezers are not required
–Significant cost savings
–Improved chance of high quality samples due to simpler process
Unfortunately, it’s not as simple as that...
Hematocrit Levels NominalNeonates (Age 0 to 1 year) 28 to 67Children (Age 1 to 12 year) 33 to 45Adult female 36 to 48Adult male 40 to 52Anaemia <33Polycythenia > 54 (adult), 68 (infants)
• Investigated different human hematocrits– Plasma removed or added as required
Effect of Hematocrit on DBS Sampling
�When spotting a fixed volume of blood, the HCT affects the size of spot derived–High HCT gives a small spot
�If a fixed diameter sub-punch is taken from the sample, a different volume of blood is sampled depending on HCT–Introduces assay bias
–Recovery and suppression may also play a role
70%45%20%
Potential Approaches to Minimising the HCT Effect for DBS Sampling
�Apply a constant volume of blood and extract the whole dried spot– Spotting solution has to be simple and capable of implementation in
busy study environment
– For clinical studies, the device has to be cheap
– Can it be guaranteed that a fixed volume has been spotted?– Puts the burden on the person collecting the sample, rather than the
bioanalyst
�Maximise recovery– Evidence that recovery greater than 80-90% for high HCT samples is
preferential, as little variance is then observed throughout the HCT range
�Alternative substrates– Evidence that the HCT effect is less pronounced on treated
substrates
Other Issues with DBS Sampling
�New technology – not fully understood–Increasing numbers of publications
�‘Push-back’ from Regulators–FDA requirement to compare dry to wet blood for a number of clinical
studies
Cross Company Consortia
�EBF (Europe)–Topic team formed for blood microsampling & DBS approaches
–Sub-teams to investigating–Hematocrit
–Stability
–Dilutions
–Use of internal standard
�IQ Consortium (USA)–Meetings with FDA to clarify required approach for submitting
clinical DBS data
–Publication of ‘white papers’
Other Approaches to Microsampling at GSK
�Aim to have the simplest possible method of collection
–Gives the best chance of a high quality sample
–Considerations of the bioanalytical laboratory are of secondary importance
�Plasma microsampling
– Essential where TK/PK is in plasma, rather than blood– Degree of blood cell association
�Blood microsampling
–Freeze whole blood sample at the point of collection
–Blood / water–Relies on both the blood sample and water diluent having accurate volumes
–Accurate volume DBS–Currently ‘road testing’ a number of cheap disposable devices–Plan to implement in 1Q13
Plasma Microsampling
�Development in collaboration with Drummond Scientific (USA)–Collect 75 µL of blood into EDTA
coated, mylar wrapped microcapillary containing thixotropicgel & self-sealing plug
–Centrifuge inverted microcapillary in 1.4 mL Micronic tube fitted with a pre-split cap
– Tube acts as convenient holder & facilitates labeling
–Dispense entire plasma sample (40 – 45 µL) into labeled screw-top tubes
–Freeze for storage & transport–Extract using method developed for
5 – 15 µL plasma
�Likely to implement in 1Q13
Whole Wet Blood Microsampling
�Minimise sample manipulation
– Better quality sample = better quality data
�Sample ca. 80 µL whole blood
�Freeze whole blood sample at the point of collection
– No addition of water, or sub-aliquotting
�Ship and store blood sample frozen
�Defrost whole blood sample at site of analysis and take 20 µL aliquot
�Add IS to sample in 10 µL water/MeOH (1:1 v/v) prior to extraction
– Preferably stable isotopically labelled IS
�Stand for 1 hr
�Crash with 100 µL MeCN
�Currently under investigation
Conclusions
�Continuing to move forwards with microsampling at GSK and at other companies
– Cross company initiatives to increase understanding and improve processes concerning DBS sampling
– Regulatory framework becoming more clear
– Advancing simple approaches for plasma microsampling and accurate spotting DBS
– Note – All approaches other than DBS require freezing of the sample