Time to Coagulation in Tubes

1. Proposed Plasma Micro-Sampling Device for Implementation in Pre-Clinical Studies Chester L Bowena, Molly Karlinseya, Hermes Licea-Pereza, Kristen Jurusika, Esaie Pierrea Jim Kenneyb and Joe Sipleb a Bioanalytical Science and Toxicokinetics, PTS DMPK, GlaxoSmithKline, King of Prussia PA b Drummond Scientific Company, Broomall PA Pre-Aliquot Feasability Using the multiple aliquot dispenser provides the possibility for pre-aliquoting into extraction tubes at collection site. Two concerns are accuracy of dispensers, but also the homogeneity of the drug concentration in plasma fixated in the capillary. An investigation was undertaken using three chemically diverse compounds. These compounds were separately spiked in fresh whole blood (at three levels), collected in the capillary, centrifuged, and aliquoted (using multiple aliquot dispenser) into two distinct tubes for extraction. Introduction The benefits of micro-sampling to preclinical environments have already been recognized throughout the pharmaceutical industry through the implementation of Dried Blood Spot (DBS) technology. However, variable hematocrit levels in animal and patient populations could have an impact on accurate spotting and analysis due to differences in blood viscosity. Advances in LC/MS/MS technology, specifically instrument sensitivity, have allowed for a decrease in sample volume, facilitating the implementation of micro-sampling approaches in bioanalysis. Generally, plasma assays are preferable in the bioanalytical community as compared to DBS, whole blood, and blood/water assays. The use of plasma micro-sampling in preclinical toxicokinetic (TK) studies could facilitate the removal of satellite animals. As a result, blood sampling from the TK animals will allow direct correlation between the drug exposure and toxicity observed in the same individual animal as well as greatly reduce the number of animals required for each study. One of the current challenges of micro-sampling is generating plasma aliquots from a small volume of blood (<100 µL). In this work, a prototype device is evaluated that will allow for isolation of a micro-volume of wet plasma (<40 µL) from whole blood (<75 µL). Collection and Isolation Micro-capillary tubes, containing anti-coagulant, are routinely used in both clinical and preclinical sampling environments. The commonly used tubes are Mylar® wrapped to facilitate safety during handling. In this investigation, whole blood was drawn into a specially designed micro-capillary separation tube containing EDTA and a thixotropic gel. One end of the micro-capillary tube was fitted with a self-sealing plug that enabled the tube to be centrifuged. Prior to centrifugation, the micro-capillary tube was inverted (plug at bottom) in a labeled 1.4 mLmicronic tube fitted with a pre-split cap. Upon centrifugation, the gel migrated based on density to form a stable barrier between the erythrocytes and plasma. • Time to Coagulation in Tubes • Due to the manufacturing process of adding the EDTA anticoagulant into the capillary tubes and inability to mix the blood in the tubes, experiments were designed to test whole blood coagulation time in the capillary tubes • Fresh human blood was placed into both untreated and EDTA-coated micro-capillary tubes immediately following collection. • Mouse blood was collected directly into the EDTA-treated micro-capillary tubes from the mouse tail vein. • Blood clotted within 15 minutes in untreated micro-capillary tubes • No visible clotting in EDTA-coated micro-capillary tubes for up to 60 minutes after collection 80 µL of blood Experimental Blood was collected from rats dosed independently with acetaminophen and moxifloxacin. Plasma concentrations were determined for samples collected using both the conventional microtainer and micro-capillary tube Conclusions • Micro-sampling of plasma using self-sealing, micro-capillary tubes and dispensers was successfully demonstrated. These prototype devices facilitated blood collection, plasma separation via centrifugation, and dispensing. • Assay performance was consistent with precision and accuracy requirements as set by regulatory standards. • Use of the Micronic sample storage tubes at 0.5 mLallowed for ease in sample manipulation. Freeze-thaw had minimal effect on the sample stored in the tubes, and lyophilizationwas not observed in the tests performed. • Micro-volume collection in rodent species has successfully been carried out with micro-capillary tubes in controlled environment. Polymer Plug Plasma Dispensing and Storage Typical assays utilize 10 µL of plasma, and small volume errors could have a major impact on accuracy. In this work, aliquots of plasma ranging in a fixed amount between 10 and 15 µL were dispensed using devices designed to interface with the micro-capillary tube. Dispensers were also used to dispense the entire plasma volume. Recovered plasma was then stored in 0.5 mLlabeledMicronic tubes with screw caps. These smaller tubes (when compared to 1.4 mL tubes) were found to be better when dealing with these micro-volumes, and are still able to be formatted into conventional automation techniques (de-cappers and Hamilton liquid handlers). • Rodent Blood Volumes • ACUC (Animal Care and Use Committee) protocols allow for 10% of the total blood volume to be collected over 24 hours • Assuming a maximum blood volume of ~11 mL, estimated for a female, 6 week old HAN rat weighing ~200 grams (i.e., the smallest rat), only ~1.1 mL of blood is available for sampling within the 24 hour period Future Directions GSK is currently undertaking a global evaluation of the proposed plasma micro-sampling device involving numerous rodent studies. Upon successful completion of evaluation, this process will be implemented for rodent studies requiring plasma collection and analysis. Acknowledgement The authors would like to acknowledge Alan Stokes (GSK Safety Assessment) for performing the acetaminophen experiments. All studies were conducted after review by the Institutional Animal Care and Use Committee at GSK (or at the institution where the work was performed, if not at GSK) and in accordance with the GSK Policy on the Care, Welfare and Treatment of Laboratory Animals.