Sponsored Content THE CAPILLARY MICROSAMPLING METHOD
Ove Jonsson and his colleagues have pioneered the use of CMS at AstraZeneca. They originally developed the technique for the collection and analysis of 25-µL blood samples in rat and dog toxicology studies, and analysis of 8-µL plasma samples prepared from 32-µL blood samples in a mouse toxicology study (Ref 2). They pushed the limits by scaling down further to enable repetitive collection of blood, plasma and serum samples from mice with CMS samples as small as 4 µL (Ref 3). These efforts were rewarded when, together with Kristian Königsson and Marie Eriksson, Ove Jonsson received the AstraZeneca global 3Rs award in 2012.
In their CMS method, a blood volume of approximately 20 µL was collected in a K2
EDTA hematocrit tube to prepare
blood and plasma samples (see figure). Approximately 16 µL of blood were also collected in a hematocrit tube without anticoagulant and set aside to allow blood coagulation to give a serum sample. An exact volume capillary was then held end-to-end with the first K2
EDTA capillary to remove
4 µL whole blood, which was stored frozen. The remaining blood in this tube was stored on ice and then centrifuged, together with the blood without coagulant to prepare plasma and serum, respectively. These hematocrit tubes were cut above the blood cell phase and exact volumes of 4 µL were removed using the same end-to-end technique. Blood, serum and plasma samples were stored frozen. Prior to analysis, the samples were thawed and flushed out of the capillaries with buffer.
Warm animal for 30’ and sample from lateral tail vein using capillary tubes ACCEPTANCE OF MICROSAMPLING
The Bioanalysis Zone (Future Science Group) recently completed a survey of researchers using microsampling in both pre-clinical and clinical studies. Respondents ranked their reasons for using microsampling in preclinical studies as follows:
1. Reduction in number of animals used 2. Ability to collect more samples per animal/subject 3. Cost saving
4. Drug exposure data in main study toxicology study animals
5. Reduced stress to animals
Microsampling was also used in clinical studies, primarily in pediatrics.
The respondents did, however, express some concerns around microsampling, including:
1. Ability to handle small volumes accurately
2. Availability of analytical techniques with sufficient sensitivity
3. Acceptance by regulators
Firstly, handling small volumes may seem daunting, but as Marie Eriksson, formerly at AstraZeneca says, “Rats and mice are the most commonly used animal in life science, and handling small sample volumes is therefore critical. Microsampling is a key technique, a technique that has to be learned but is straight-forward for anyone experienced in working with laboratory animals.”
20 µL blood + anticoagulant
16 µL blood -anticoagulant
Prepare plasma and serum and transfer to exact volume 4 µL capillary tubes
4 µL blood
Seal and store to separate plasma
Centrifuge
Seal and store to coagulate
The second concern is being met by the increasing sensitivity of analytical techniques. The case studies described here show how one analytical method – nanoliter-scale immunoassay – complements the microsampling method perfectly, enabling studies to be made that would have been impossible a few years ago.
Finally, regulatory acceptance will come once there is confidence in the science, supported by studies such as those summarized here (Ref 5).
Cut capillary above blood phase
4 µL plasma Store capillary tubes at -70˚ C
Example of microsampling using CMS to prepare blood, plasma and serum samples, based on Ref 3.
4 µL serum
The benefits of microsampling were clearly illustrated in a report on toxicity testing at AstraZeneca, published by the Swedish Toxicology Sciences Research Center (SWETOX; Ref 6) in collaboration with former AstraZeneca employees in July 2014. Data were analyzed from 36 projects on animal use reduction at AstraZeneca, showing that substantial reductions could be achieved through project coordination, improved study design and, most importantly, method development. Microsampling was an important contribution in method development, made possible by improvements in analytical capacity.
Through these reduction projects, AstraZeneca realized an overall reduction in animal use by 53%. An equivalent reduction at each of the top pharmaceutical companies could reduce the total number of research rats used annually worldwide by as many as 150,000 animals.
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