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Enter an Australian post code for delivery estimate. When using small volume tubes, particular attention must be paid to blood collection practices. The rapid transfer of blood to the anticoagulant tubes is important to limit blood clotting and contamination of citrate tubes. The preferred order of tube filling from the syringe to the tubes, to limit blood clotting, is the following: Citrated tubes should be filled first, followed by EDTA, heparin, SST, and nonanticoagulant tubes filled last.
Tubes must be capped and inverted immediately several times. It is ideal to have one technician designated for blood collection and tube filling, and a second technician for capping and inverting tubes. These are useful for TK samples but will not be useful for CP testing because such small volumes are very difficult to analyze without diluting the collected blood.
Based on survey responses, there appear to be limited opportunities for sample volume reduction for CP evaluations.
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However, it is possible to collect the lower range of required volumes Table 1 , as 0. While conventional analytic TK methods have required up to 1 mL blood per time point, recent analytic advances have significantly reduced volume requirements for TK analysis, opening the door to the use of microsampling for TK blood collections.
The assay sensitivity has to be taken into consideration for appropriate volume selection. Though not widely adopted, microsampling for TK or PK and pharmacodynamic PD assessments has proven to be a viable approach for refinement and reduction of animal use in safety studies. Use of microsampling was very limited in definitive GLP studies done for general toxicity, safety pharmacology, or genetics toxicology.
The largest influence on rodent numbers has the collection of samples for TK analysis. Separate TK cohorts are generally preferred in regulatory studies to ensure tolerability, prevent jeopardizing toxicity endpoints, and maximize the ability to detect minimal CP changes. Serial TK sampling is best to assess individual animal changes over time but requires collection of large cumulative blood volumes from each animal, usually necessitating separate TK satellite animals.
Composite sparse sampling reduces the total volume of blood collected in each animal but requires more animals. In addition to reducing animal use and compound requirement, this approach enables direct determination of exposure in main study toxicity animals, allowing correlation of exposure and toxicity in individual animals refinement.
This becomes especially important when drug exposure or distribution is not consistent across all animals in a group. Several researchers have evaluated the impact of TK collections on CP variables in rats, and have reported minimal effects that were generally due to repeated handling mild stress leukogram or prior blood collection lower RBC mass, increased reticulocyte counts. Furthermore, this blood volume could not be obtained consistently for all time points and sampling was frequently associated with hematomas at the blood sampling site.
Collection of blood volumes up to 1. The effect of TK collections on hematology testing in main study animals can be minimized by sampling for hematology prior to TK collections and sampling for biochemistry and coagulation after TK collections prior to or at necropsy. In order to limit blood volume requirements and eliminate the need for satellite TK groups, the number and frequency of TK blood collections may be reduced. However, the number of CP time points required for a given study can be minimized using information gained in earlier, preliminary studies.
Another approach to allow for sampling of larger volumes while using fewer animals is to use larger animals. Some may consider using only male animals, which have a larger body weight and blood volume compared to female animals, when no sex differences in test article effects are anticipated. Similarly, increasing the age of rats at study initiation allows collection of large volumes during the first week of study, when characterization of the initial exposure profile or PD response requires repeat blood sampling over the first one to 2 weeks following first dose administration.
Initiating dosing of rats at 12 weeks instead of 6 weeks of age will allow for collection of 1. The difference in blood volume between 6 and 12 weeks is less evident in mice.
In this species, separate cohorts of animals are commonly used in order to obtain a sufficient quantity of blood for the measurement of all hematology and biochemistry variables. Coagulation profiles are rarely done in mouse studies or should be limited to terminal blood collection. Reducing the number of variables evaluated within the study is of limited utility as hematologic endpoints are all measured during the same analysis with a predetermined blood volume. Clinical biochemistry endpoints may be prioritized but most should be measured in order to avoid jeopardizing the scientific integrity of the study.
Some may consider limiting the required blood volume to the minimum volumes needed by the analyzers, but collecting enough blood to allow potential repeats for the verification of initial data is a core foundation for CP assessment.
In unusual circumstances, when the risk generated by nonverified analysis is accepted and not judged critical, it could be possible to decrease the blood volume requirements to that necessary for only one analysis. In these instances, a blood smear should be prepared before hematology analysis to verify any abnormality by microscopic evaluation, and the use of properly validated and quality controlled instruments should be ensured. Irrespective of the success of microsampling for TK assessments, adaptation to CP testing is not currently feasible, primarily because of analytic requirements for larger volumes.
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As discussed, the sample volumes required by current automated analyzers for CP panels are much higher than those collected via microsampling. Because of the limited feasibility for microsampling and sample dilution in CP, efforts to reduce animal use have been focused on microsampling for TK and study design modifications.
There are opportunities for blood volume reductions, especially for nonrodent species because blood volumes requested often exceeded the volumes required for analysis. With most standard analyzers commonly being used in industry, 0. Sample pooling is not considered adequate because it precludes investigation of changes occurring in individual animals in preclinical studies. Sample dilution as a means to reduce blood volume requirement for CP testing is not considered adequate in preclinical studies due to the introduction of preanalytic and analytic errors associated with this practice.
An appropriate phlebotomy site should be determined based on blood volume requirements and technical expertise of the staff. Jugular vein and sublingual vein collection are appropriate for repeated survival blood collections in rodents. Microsampling is not being utilized, nor is it considered suitable for CP testing in toxicity studies because it does not provide sufficient volume to accommodate all required CP tests given current analyzer requirements. Approaches such as reducing the frequency of the CP evaluations or the number of variables evaluated within a study, and limiting the blood volume collected to the minimum volume required with no possibility of repeat analysis, are of limited interest because such practices may jeopardize the generation of critical study data.
Avoid collection of maximal blood volume allowed by IACUC for hematology collections, particularly for studies with potential hematotoxicity. For mouse studies, use separate subsets of animals for hematology and clinical biochemistry testing, and establish a priority list for clinical biochemistry analytes to ensure determination of critical tests in the event of short samples.
When possible, eliminate coagulation testing from mouse studies. In conclusion, although it is possible to make modest reductions in blood volume requirements for CP testing in toxicologic studies, current regulatory and analytical methods are not amenable to the application of microsampling and preclude extensive modification of current testing paradigms. Major opportunities to reduce blood volume requirements for CP testing rely on optimization of blood collection and analytic methods and alterations in study design.
Blood is taken from a small triangle just under the scapula. Up to 0. The animal is restrained with one hand placed around the thorax, the forelimbs are grabbed with the technician's thumb and middle finger placed at the level of the biceps and pushed toward the back of the animal. The head is tilted at an angle in the head cap, which makes the site of sampling prominent. The collection is done by inserting the needle 1—3 mm deep under the clavicle in the direction of the muzzle. With the animal in a supine position grasping the dorsal cervical skin scruff of the neck , the mouth will open and 2 sublingual veins located at the base of the tongue on either side of midline will be visible.
The mouth should be rinsed and dried with gauze prior to blood collection to minimize contamination of the sample with saliva. After the desired blood is obtained, pressure is applied to the tongue using cotton or gauze and bleeding will generally cease in 20—30 seconds. In mice, the sublingual vein is caudally located and more difficult to visualize while guinea pigs do not have a sublingual vein. While holding the distal portion of the tail in one hand, the needle is inserted at an oblique angle to the vein. When using a catheter, the tubing is removed to prevent clotting.
The blood is allowed to drip into 0. Abdominal aorta and vena cava are the most common sites for terminal collection in rodents. Blood is sampled from these sites under anesthesia and after opening of the abdominal cavity. Animals are then euthanized before necropsy. This type of blood collection allows collection of larger blood volumes as partial exsanguinations may be performed. A sample of 10—15 mL blood can be obtained from rats, and 0. Cardiac puncture is only carried out under deep terminal anesthesia, or on euthanized animals for any species.
Blood samples are taken from the heart, preferably the left ventricle, which can be accessed either via the left side of the chest, through the diaphragm, from the top of the sternum or by performing a thoracotomy. Alternatively blood can be sampled from the right ventricle for venous sampling. Note : As discussed earlier, while this method can be used for TK studies, it is not suitable and cannot be recommended for clinical pathology testing.
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Tail vein capillary sampling is suitable for capillary tube collection in mice and rats. The tail is incised with a razor blade after being dipped in warm water, and it is squeezed at the base of the cut for blood collection. The Pasteur pipette allows collecting larger volumes, up to 0. Submandibular vein capillary sampling does not necessitate anesthesia and can be used as collection site for mice. Volume 45 , Issue 4. If you do not receive an email within 10 minutes, your email address may not be registered, and you may need to create a new Wiley Online Library account.
If the address matches an existing account you will receive an email with instructions to retrieve your username. Veterinary Clinical Pathology Volume 45, Issue 4. Position Statements and Special Reports developed by the ASVCP provide current information on topics in veterinary clinical pathology that are important to the veterinary community. The procedure for submitting statements is detailed at www. The final draft is edited prior to publication in Veterinary Clinical Pathology. Tools Request permission Export citation Add to favorites Track citation. Share Give access Share full text access.
Share full text access. Please review our Terms and Conditions of Use and check box below to share full-text version of article. Abstract In preclinical safety assessment, blood volume requirements for various endpoints pose a major challenge. Introduction There is widespread interest in improving animal welfare in toxicologic research, as advocated in the 3Rs principles of reduction, refinement, and replacement of animal use in research and teaching. Regulatory guidelines In preclinical safety assessment, blood samples are collected for evaluation of TK and CP variables, and other biomarkers.
Instrument and Analytic Volume Requirements In order to define open questions regarding current blood collection practices, volume requirements, instrumentation, and analytic integrity of sample dilution, the Regulatory Affairs Committee RAC of the American Society for Veterinary Clinical Pathology ASVCP surveyed veterinary clinical pathologists and other laboratory professionals active in preclinical development and safety studies.
Hematology According to the survey respondents, minimum blood volumes requested for a single hematology evaluation were largely driven by analyzer requirements. Clinical biochemistry Similar to the minimum volume requirements for hematology testing, minimum blood volumes requested for biochemistry testing were largely driven by analyzer requirements and the need for obtaining sufficient reserve sample for repeat analysis and technical limitations. Coagulation Among standard CP panels, coagulation testing required the highest sample volumes. Sample Pooling Some investigators may suggest economizing blood by pooling samples from individual animals.
Phlebotomy Volumes, Sites, and Methods of Collection The survey requested information regarding which phlebotomy sites are used for each of the major laboratory animal species rat, mouse, NHP, and dog during both interim and terminal collections, and conditions where blood volume may be limited eg, interim or survival intervals. Rodents rats and mice Rodent terminal collections by needle and syringe or catheter In rodents, large volumes of blood can be drawn from anesthetized animals at study termination, via large vessels using a needle and syringe or a catheter. Rodent nonterminal collections by needle and syringe or catheter For nonterminal interim or survival collections, blood sampling using a needle and a syringe or a catheter is the method of choice because it allows for rapid collection of large volumes.
Rodent capillary or pipette blood collection Blood may be collected using capillary tubes or Pasteur pipettes via a skin laceration or nick of mucosal tissue. General considerations for low volume blood collections Reducing preanalytic variables Preanalytic variables may influence small blood volume results, particularly in mice and rats as blood sampling difficulties are more frequently encountered in these species. Opportunities for Reducing Animal Use Based on survey responses, there appear to be limited opportunities for sample volume reduction for CP evaluations. Modifying study designs to optimize CP collections In order to limit blood volume requirements and eliminate the need for satellite TK groups, the number and frequency of TK blood collections may be reduced.
Conclusion and Points to Consider Irrespective of the success of microsampling for TK assessments, adaptation to CP testing is not currently feasible, primarily because of analytic requirements for larger volumes. Sublingual blood draws With the animal in a supine position grasping the dorsal cervical skin scruff of the neck , the mouth will open and 2 sublingual veins located at the base of the tongue on either side of midline will be visible. Terminal Blood Collection Abdominal aorta and vena cava are the most common sites for terminal collection in rodents.
Accessed May 10, Google Scholar. Crossref PubMed Google Scholar. PubMed Google Scholar. Crossref Google Scholar. Citing Literature. Volume 45 , Issue 4 December Pages Figures References Related Information. Close Figure Viewer. Browse All Figures Return to Figure. Previous Figure Next Figure.
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Email or Customer ID. Forgot password? Old Password. New Password. Password Changed Successfully Your password has been changed. Returning user. Request Username Can't sign in? Forgot your username? Enter your email address below and we will send you your username. Regulatory guidelines and restrictions on blood collection. Nonrodent nonhuman primate and dog. Rodent terminal collections by needle and syringe or catheter.
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Rodent nonterminal collections by needle and syringe or catheter. Rodent capillary or pipette blood collection. General considerations for low volume blood collections. Reducing preanalytic variables. Considerations on the use of small collection tubes. Microsampling for toxicokinetic TK analysis.
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