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

Acknowledgements

�Philip Denniff

�Chet Bowen

�Olga Woolmer

�Christopher Evans

�Drummond Scientific

Storing microvolumes of plasma or blood

�0.5 mL micronic tube with either screw or push cap�Compatible with all current automation�Tubes have a tappered bottom�Available in non-coded, alphanumeric and 2D coded (Tracker Data-Matrix or TraXis)

80 µL of blood