Methodology Spooner, Denniff, Michielsen et al. Key terms
Hematocrit: Ratio of the volume of red blood cells to the volume of whole blood.
Volumetric absorbtive microsampler: A technique for the collection of dried biological samples for quantitative bioanalysis by absorbing a fixed accurate volume of blood onto an absorbent tip. The commercial name for these samplers is Mitra™.
does not spread homogenously on the DBS material (substrate). Furthermore, the hematocrit (HCT) of the blood affects its viscosity and so gives rise to dif- ferent-sized DBS spots, with high-HCT blood giving smaller blood spots and low-HCT blood giving larger spots. Thus, the volume of blood in a fixed-diameter subpunch taken from these samples is difficult to determine, resulting in lower-quality drug concentra- tion data for samples, particularly in which the blood HCT varies markedly from that of the control blood used to prepare the calibrant and quality control (QC) samples. A number of laboratories have published manuscripts
describing novel approaches to overcoming these issues regarding HCT and homogeneity with DBS [22–25]. Unfortunately, these approaches rely on spotting an accurate volume of blood, measuring another blood component or are not readily commercially avail- able. Thus, their implementation for day-to-day study support may be impractical. A novel dried blood sampler, termed the volumetric absorbtive microsampler
(VAMS), has been
designed in order to deliver the benefits of DBS sampling while overcoming the issues associated with HCT and homogeneity and also enabling further simplification of the sample collection and processing/extraction work- flows [26]. The VAMS consists of an absorbent polymeric tip designed to take up a fixed volume of blood (nomi- nally 10 μl) by capillary action. The tip is attached to a handle by a plastic pin (Figure 1). The handle has been designed to be ergonomic to hold and has fins to help locate it within 96-well blocks during extraction and to minimize the possibility of the sampler tip coming into contact with surfaces during storage and shipping. The sampler is filled by holding the handle part of the sam- pler and dipping only the leading surface of the tip into a pool of blood and allowing it to fill. The tip of the sam- pler must not be completely submerged into the blood sampler, as this may cause overfilling. When the tip has turned completely red, it is full, which takes 2–5 s, mak- ing the device self-indicating. The last part of the tip to turn red is the shoulder. In addition, the sampler is engineered to fit a standard laboratory air-displacement pipette or automated liquid handler in order to allow for automation during extraction of the blood sample.
654 Bioanalysis (2015) 7(6) This article describes results from a cross-company
and -laboratory gravimetric experiment to determine the volume of blood absorbed by the sampler at vari- ous blood HCT values (∼20, 45 and 65%). Further- more, in order to convert the weights of the blood absorbed by the sampler into volumes, the density of the blood at each HCT was determined by each laboratory.
Materials & methods Equipment & reagents VAMSs were supplied by Phenomenex, Inc. (CA, USA; exclusive distributor for the Mitra™ micro- sampler, manufactured by Neotryx, LLC, CA, USA). Control EDTA blood was obtained from the fol- lowing suppliers in accordance with each company’s current policies on informed consent and ethical approval: human blood was obtained from: Glaxo- SmithKline (GSK Stevenage, UK) from in-house facilities and used within 7 h of draw; Merck from Biological Specialties (PA, USA) and used within 10 h of draw; Janssen from the Clinical Pharmacol- ogy Unit (Belgium) and in-house and used within 4 h of draw; and Sanofi from Biopredic Interna- tional (Saint-Gregoire, France) and used within 48 h of draw. Rat blood was obtained from Bioreclamation (Brussels, Belgium) and used within 2 weeks of draw (Bristol-Meyers Squibb [BMS]) or from in-house facilities used within 7 h of draw (AstraZeneca [AZ]). Blood samples with different nominal HCT values of 20, 45 and 65% (and additionally at 60 and 69% for two laboratories) were prepared at each participating laboratory by determining the HCT of the control blood by centrifugation and then either removing the appropriate volumes of plasma from centrifuged blood or adding plasma to blood that had not been centrifuged, followed by gentle mixing. The HCT of the modified blood samples was then confirmed using centrifugation [19]. All experimental sites used microbalances that mea-
sured to at least five decimal places of a gram. GSK used a Sartorius R200D, Janssen used a Sartorius CPA225D, BMS used a Sartorius Cubis Precision Lab Balance, AZ used a Sartorius MC210P, Merck used a Mettler Toledo SAG 285 and Sanofi used a Mettler Toledo AT261. The pipettes used were Gilson Micro- man M10 (GSK), Eppendorf Reference (Janssen), Biohit mline (10–100 μl; BMS), Gilson Microman M25 (AZ), Eppendorf positive displacement repeaters equipped with a 0.1-ml ‘Combitip’ (Merck) and Bio- hit Proline (0.5–10 μl; Sanofi). Scintillation vials were obtained from Thermo Fisher (20 ml: GSK and BMS; 7 ml: Merck), Fiolax (20 ml: Janssen) and Perkin Elmer (20 ml: AZ and Sanofi).
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