GentleSharp - a more humane system for small volume blood sampling in PK and toxicology research

Summary | Background | Current state of the art | What could your solution be used for? Collaboration | 3Rs impact


Summary

The GentleSharp™ system improves blood sampling success by using backward and forward micro-oscillations to help the needle puncture and glide through tissue more easily. This reduces animal discomfort, decreases stress and enables greater needle control and up to 15% higher collection success. Blood collected from animals using GentleSharp™ had reduced plasma corticosterone following serial collections over several weeks, and animals exhibited reduced movement and vocalizations during sampling procedures. Blood collections using the GentleSharp™ system required fewer needle insertions but resulted in increased sampling blood mass.


Background

Blood sampling is one of the more common practices in biomedical research. A number of techniques and routes for obtaining blood samples exist (7;17), some routes require/recommend anaesthesia (jugular, retro-orbital), while others do not (tail vein/artery, saphenous vein, submandibular vein). Sampling stress, including painful needle punctures, can increase stress hormone levels potentially confounding subsequent blood chemistry analysis (5;15) resulting in increased variability in the data obtained. Larger group sizes are often required to overcome this. The pain is thought to be produced by compression of the outer layers of skin which activates the free nerve endings of the dermis. Methods that can reduce the penetration force such as smaller needle diameters and sharper tips seem to reduce the pain and discomfort (8). More specific to needle oscillation, research has demonstrated that oscillating needles during insertion leads to reductions in both puncture and friction forces (12;13). The increased needle velocity from oscillation results in decreased tissue deformation, energy absorbed, penetration force, and tissue damage (10; 11;16). The reduction in force and tissue deformation from the increased rate of needle insertion is especially significant in tissues with high water content such as soft biological tissue (3). In addition to reducing the forces associated with cutting into tissue, research has also shown that needle oscillation during insertion reduces the frictional forces between the needle and surrounding tissues (9).


Current state of the art

Guidelines are established for the frequency and amount of blood that can be collected from a single animal (20; 21). Additionally, repeated or serial sampling in a single animal is often difficult, especially when cannulation is not an option. This is especially true for study designs requiring multiple samples within a shorter period of time, possibly due to tissue damage or stress-induced vasoconstriction. To circumvent this issue, researchers often resort to cross-sectional study designs requiring large numbers of animals to be used at different time points to obtain the required blood samples and volumes necessary for analysis (2). This approach can be expensive, uses large numbers of animals, and limits the data that can be obtained, as the data is derived from multiple animals, rather than a single animal tracked over time (6). The practice of microsampling is growing in popularity as samples can be obtained from the same animal longitudinally and technology has improved such that small blood volumes (generally a microsample is defined as < 50 ul) (14; 18) are adequate for many analyses. Smaller samples are more readily collected from each animal with minimal restraint, lowering the potential of tissue damage (1) and reducing stress responses while increasing the number of samples that can be obtained from each animal (19). A number of approaches exist for microsampling (4), which can be facilitated with GentleSharpTM by decreasing the number of needle insertions required to yield blood and increasing sampling success.  This is particularly advantageous when samples of up to 200 µl are required (generally a microsample is defined as < 50ul) (13, 14). This enables longitudinal study designs where the same animal can be sampled (low volume/microsampling) at multiple time points, decreasing variability in the data and the total number of animals needed per study. The stress from handling the animals is unavoidable, but by increasing sampling success and reducing the number of needle insertions for each collection point, this allows the animals less exposure to trauma. 


What could your Solution be used for?

GentleSharp™ was designed to help address challenges of animal blood sampling. It allows for serial low-volume blood sampling in rats and mice. Currently, GentleSharp™ is designed for low-volume sampling with collection via a capillary tube (appropriate for research collecting pharmacokinetic and pharmacodynamic data). (Current studied protocols have included a short four minute tail warming pre-treatment prior to sampling attempts.) Further expansion of the product line, will allow for larger volume blood collections, from larger animals, and for injection capability.


Need for collaboration

GentleSharp™ was conceived, designed, and developed by Actuated Medical, Inc. – experts in the design and development of next-generation, FDA-compliant medical devices. Formed in 2006, our innovations have spawned a new generation of medical devices that incorporate Innovative Motion® technologies to improve clinical care and patient outcomes. We focus on state-of-the art, minimally invasive instruments for clearing occlusions, penetrating tissue, and enabling the emerging MRI-guided surgical procedure industry

GentleSharp, Inc., is seeking partners able to provide a greater species diversity in animal subjects undergoing low volume blood sampling (< 50 µl); of particular interest would be the same population of animals undergoing repeated samplings. GentleSharp™ has been used on both rats and mice; however, we wish to expand this to rabbits, guinea pigs, ferrets, etc. We have completed studies from blood collections from the tail artery (mouse), tail veins (rats) and submandibular (mouse).  We aim to expand our current number of studies, especially with alternate target collection sites, such as saphenous and submandibular in multiple species. Our goals are twofold. First, we want to validate the technology across a wider variety of species and collection sites. More importantly, we want to broaden the usage of this technology to maximise the refinement and reduction benefits.


3Rs impact assessment

GentleSharp™ improves blood sampling outcomes with both rats and mice, enabling the acquisition of blood samples while eliciting a reduced stress response, benefitting studies conducted with either rats or mice (19).

  • Refinement: GentleSharpTM reduces the levels of plasma corticosterone in rats (up to 65% reduction during serial sampling) and a trend for reduction in mice. Moreover rats exhibited reduced behavioural indications of stress (reduced animal movement and vocalization) when GentleSharp™ was ON vs. OFF. By providing a mechanism to improve on tail sampling, GentleSharp™ can replace crude methods such as tail snipping (in mice), especially when cannulation is not a viable option. 
  • Reduction: GentleSharpTM reduces the individual variance in corticosterone levels by up to 40% over the study span in the animals. The improved reproducibility afforded by GentleSharpTM could allow for a reduction in the number of animals necessary in future studies. This would save the cost of the additional animals, and also reduce the time required to complete the analysis if less samples had to be processed. Moreover, by having the ability to serially sample the same animal, the researcher can track an individual animal over time, allowing more complete data collection.

References

  1. Burnett JE (2011). Dried blood spot sampling: practical considerations and recommendation for use with preclinical studies. Bioanalysis 3:1099-107.
  2. Cano P et al. (2008). Effect of aging on 24-hour pattern of stress hormones and leptin in rats. Life Sci 83:142-148.
  3. Chan KK et al. (1985). The Mode of Action of Surgical Tissue Removing Devices. IEEE 1985 Ultrasonics Symposium; p. 855-9.
  4. Chapman K et al. (2014). Overcoming the barriers to the uptake of nonclinical microsampling in regulatory safety studies. Drug Discovery Today 19:528-532.
  5. Fitzner Toft M et al. (2006). The impact of different blood sampling methods on laboratory rats under different types of anaesthesia. Lab Anim 40:261-274.
  6. Hofer SM, Flaherty BP, Hoffman L (2006). Cross-Sectional Analysis of Time-Dependent Data: Mean-Induced Association in Age-Heterogeneous Samples and an Alternative Method Based on Sequential Narrow Age-Cohort Samples. Multivariate Behavioral Research 41:165-187.
  7. Hoff J (2000). Methods of blood collection in the mouse. Lab Animal 29:47-53.
  8. Kaushik S et al. (2001). Lack of pain associated with microfabricated microneedles. Anesth Analg 92:502-504.
  9. Khalaji I et al. (2013). Analysis of needle-tissue friction during vibration-assisted needle insertion. Intelligent Robots and Systems (IROS). IEEE/RSJ International Conference p. 4099-104.
  10. Mahvash M, Dupont P (2009). Fast needle insertion to minimize tissue deformation and damage. IEEE International Conference on Robotics and Automation: 3097-102.
  11. Mahvash M, Dupont P (2009). Mechanics of Dynamic Needle Insertion into a Biological Material. IEEE Trans Biomed Eng.
  12. Marx JA et al. (2008). The Effect of Vibration on the Needle Dynamics of Sclerotherapy. Australasian College of Phlebology 12th Annual Scientific Meeting; Gold Coast, Australia.
  13. Muralidharan K (2007). Mechanics of soft tissue penetration by a vibrating needle. Baltimore, MD: University of Maryland Baltimore County.
  14. Powles-Glover N et al. (2014). Assessment of toxicological effects of blood microsampling in the vehicle dosed adult rat. Reg Tox Pharm 68:325-31.
  15. Vahl TP et al. (2005). Comparative analysis of ACTH and corticosterone sampling methods in rats. Am J Physiol Endocrinol Metab 289:E823-828.
  16. van Gerwen DJ, Dankelman J, van den Dobbelsteen JJ (2012). Needle-tissue interaction forces--a survey of experimental data. Med Eng Phys. 34:665-80.
  17. Waynforth H (1998). Collection of Blood Samples (Rat, Mouse, Guinea Pig, Rabbit). GOOD PRACTICE GUIDELINES 1:1-4.
  18. Bioanalysis Zone. Microsampling: the road ahead; http://www.bioanalysis-zone.com/_img/pics/pdf_1409049724.pdf.
  19. GentleSharp Ebook of Supporting Research: http://www.gentlesharp.com/feature/research-ebook/
  20. Guidelines for Survival Bleeding of Mice and Rats. National Institutes of Health. oacu.od.nih.gov/ARAC/documents/Rodent_Bleeding.pdf  
  21. NC3Rs blood sampling resource: https://www.nc3rs.org.uk/our-resources/blood-sampling

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