General principles

Blood sampling can be stressful for laboratory animals because of the handling, restraint, anaesthesia or discomfort associated with a particular technique. It is in the interests of good science, as well as of animal welfare, that stress should be kept to a minimum. Adherence to the following general principles will help to ensure that this is the case.

Licence authority, training and competency

Project and personal licence authority

  • In the UK, blood sampling is considered a scientific procedure when the sample is taken for a scientific purpose. Although removal of blood can itself have adverse consequences to the animal, the pain caused by the method of sampling (i.e. insertion of the needle into the skin) is deemed to exceed the level above which work needs to be regulated by the ASPA.
  • A project licence should detail the site, volume and frequency of blood sampling. These should be justified in terms of the science and animal welfare.
  • A personal licence is also required for the person taking the blood sample. Blood collection techniques can only be observed and not practised before applying for a personal licence. Once a personal licence is obtained, blood collection can only be carried out under supervision until the new licensee is deemed competent in the particular technique/s.

Training and competency

  • Competence in animal handling and restraint is an essential pre-requisite to blood sampling. Please see our dedicated resource on handling and restraint.     
  • Training should be done by appropriate, competent trainers so that the most refined and up to date methods are passed on. 
  • Criteria for competency should be defined for each technique. For example, competency in blood sampling from the canine cephalic vein would involve demonstrating:
    • Knowledge of the technique (e.g. suitability for purpose, appropriate needle size, permissible collection volumes, potential adverse effects and how to cope with them, how to handle the blood sample once taken)
    • The ability to restrain the dog calmly
    • The ability to suitably prepare the sampling site - including hair removal and skin preparation in a way that does not distress the dog or damage the skin
    • Accurate localisation and good dilation of the vessel
    • Insertion of the needle without causing distress to the dog
    • Removal of a non-haemolysed blood sample at an appropriate speed without causing bruising
  • Before attempting to perform any techniques, trainers should discuss with trainees the expectations regarding competency in the particular technique and how the skills will be assessed.  Factors which will determine competency include assessment of protocol compliance, attitude and empathy and consideration for health and safety as well as competence in the practical skills required for blood sample collection. 
  • The amount of training and practice required to achieve a given level of competence in a particular technique varies from individual to individual depending on, for example, manual dexterity, prior experience, attitude, and the skills of the instructor. Retraining or additional supervision is necessary if a technique is not conducted routinely and individuals should be encouraged to ask for help where necessary.
  • Inexperienced persons should first examine dead animals (euthanised for another purpose) to learn the relevant anatomy and thereby avoid having to make repeated unsuccessful entries when trying to locate a blood vessel. Use should be made of demonstration and instruction videos, as well as inanimate objects (e.g. oranges) and imitation training aids/veterinary simulators (e.g. CurVet™ rat model, UC Davis canine head and foreleg models to gain familiarity in handling and using needles and syringes, before carrying out any work on live animals. Observing experienced personnel will also help in learning the technique.

Practical considerations

  • Prepare ahead of time with labelled collection tubes and any necessary equipment for processing samples (e.g. ice, tube roller/mixer, centrifuges etc). This is particularly important for procedures such as pharmacokinetic studies where multiple blood samples are taken in close succession.    
  • Arrange any required transport/courier services in advance.   
  • Plan ahead to ensure sufficient staff resource is available to assist with the sampling procedure. This is particularly important, for example, if manual restraint rather than a restraint device is to be used or if large groups of animals are to be sampled. 

Equipment

Vacutainers

  • To prevent excess vacuuming on a syringe, consider the use of vacutainer blood collection tubes, which create a vacuum seal inside the tube to facilitate the draw of a pre-determined volume of blood. This reduces the likelihood of haematoma formation, particularly in larger animals such as dogs and primates. 
  • An advantage of using a vacutainer is that the blood does not need to be transferred from a syringe to a collection tube. In addition, vacutainers may contain additives to stabilize and preserve the blood sample.

Warming boxes

  • Blood sampling of rodents may require the animals to be warmed to dilate blood vessels prior to sampling. 
  • Best practice is to thermostatically control the temperature at no more than body heat. A timer should be used to ensure that the animal is warmed for no more than 10 minutes during which they should be kept under constant observation to prevent hyperthermia (indicated by breathing more rapidly, panting or salivating).
  • Warming boxes should not use a radiant source and temperatures greater than body heat for a shorter period are not recommended due to the risk of hyperthermia. The warming box should be calibrated regularly.
  • Warming methods are contra-indicated for pregnant animals or in circumstances where there are significant adverse effects (e.g. where the animals have reached or are nearing the humane endpoint of the study).

Restraint tubes

  • Where a rodent restraint tube is used, it should be appropriate for the size of the animal in order to avoid damage to the tail, testes and limbs.
  • All forms of restraining equipment should be frequently washed to prevent pheromonally-induced stress or cross-infection.

Aseptic technique

  • Hair around the sample collection site should be clipped and the skin aseptically prepared with a water-based chlorhexidine solution (or similar). 
  • Shaving with a scalpel blade is not recommended as it removes the epidermal layers of the skin. Hair removal cream may be considered for areas where clippers are difficult to use and in these circumstances, care should be taken to ensure that the animal does not lick the cream off.
  • Needles should always be single-use to avoid them becoming contaminated. Blunt and used needles should be disposed of directly into sharps containers.  

Venepuncture

Handling and restraint

  • Please see our dedicated resource on handling and restraint.
  • Firm, empathetic handling is very important, as is the time required to withdraw the blood sample. Both these parameters can affect the degree of stress for the animal and consequently the quality of the sample and research data.
  • The animal should be restrained by an experienced person (preferably one known to the animal, especially for larger laboratory animal species). The correct level of restraint is that which allows a satisfactory sample to be taken at the first attempt but which does not cause the animal to become unnecessarily distressed.
  • Inanimate restrainers can be used, although these may not always be the best method for individual animals. Manual restraint facilitates recognition of distress more effectively. Furthermore, restraint may not be needed if refined techniques such as, for example, positive reinforcement training or microsampling are used.
  • A vein will collapse if a sample is taken too quickly, so care should be taken to ensure that blood is withdrawn at an appropriate speed.
  • Depending on the species, consideration should be given to offering a reward after each bleed.

Needle size

  • A sterile needle (or lancet) should be used to puncture the skin and underlying blood vessel.
  • The size of the needle (length and bore) is very important.
  • It is recommended to use a needle with sufficient diameter to ensure rapid blood withdrawal without collapsing the vein, within the constraint of avoiding haematoma (i.e. the bore should be just less than the diameter of the vessel).
  • As superficial blood vessels lie close to the skin surface, needles used to sample from these veins should be kept to a minimum length. This will ensure the best possible control of the needle during insertion, reducing the risk of injury to the animal.
  • Recommendations on appropriate needle size are given for specific techniques featured within these pages. A maximum gauge of 25G for rodents and 21G for dogs is recommended.

Site and localisation of the vein

  • Choose a site that is fit for purpose and one which causes minimal stress to the animal. For example, if blood sampling after an intravenous dosing procedure, then consider whether it would be possible to sample from another vein.
  • Note that samples taken from different sites may have differences in biochemical / haematological values. Time should be spent accurately locating and dilating the vein before puncturing the vessel.
  • It is recommended that no more than three attempts should be made to collect a blood sample by the venepuncture route. If attempts to collect blood are unsuccessful, the animal should be left to recover and sampling attempted again at a later time and preferably by a different individual.

Dilation of the vein

  • In conscious rodents, blood can be more easily obtained if the animal (or part of the animal the sample is taken from, e.g. tail) is warmed first following best practice.  
  • In anaesthetised animals, vasodilation can occur as a result of the anaesthetic. Anaesthetised animals may not, therefore, need warming so consideration should be given to bleeding when animals are anaesthetised for another purpose.
  • Anaesthesia is not normally necessary for venous access, since the associated stress would probably be greater than the discomfort of a needle prick or of a puncture with a lance.
  • The use of topical local anaesthetic (e.g. EMLA) provides effective analgesia to prevent discomfort and pain on venepuncture; however 15-30 minutes contact time is required for these compounds to have full effect. To prevent animals licking the cream off once it is applied, the area can be covered with an occlusive dressing. In small rodents, this presents practical difficulties. 
  • Veterinary advice should be taken on the most appropriate anaesthetic. Some anaesthetics or components of anaesthetic mixtures (e.g. medetomidine or xylazine) cause vasoconstriction and so should be avoided.

Potential adverse effects

Potential problems that may arise as a consequence of poor sampling technique include stress, haemorrhage, bruising, thrombosis, infection at the site of needle entry, phlebitis, scarring and nerve damage. Advice on treatment for adverse effects should be sought from the Named Veterinary Surgeon.

  • Haemorrhage due to poor haemostasis is not a common problem, unless the animal has a clotting defect, and in some cases gentle continuous pressure applied for several minutes is all that is needed to stop the bleeding. Longer compression of the puncture site may be needed to stop bleeding following arterial sampling.
  • 'Bruising' is due to subcutaneous bleeding at the time of venpuncture or after the animal has been placed in its cage or pen, when the site might be aggravated by the animal itself through licking or rubbing. The animal should be checked after approximately 30 minutes and, if necessary, appropriate action taken (e.g. consult the Named Veterinary Surgeon).
  • Thrombosis (clotting) and phlebitis (inflammation of the vein) are usually caused by failure to employ aseptic technique or leaking of an irritant substance (e.g. alcohol-based chemicals) around the vein. Occasionally they can result from self-mutilation.

Arterial puncture

The main reason for collecting blood from arteries is that large samples can be obtained rapidly and relatively easily. Many of the principles described above for venepuncture also apply to arterial puncture.

Cannulation

Cannulation is an important technique for removal of blood because it reduces the stress of multiple sampling associated with, for example, repeated restraint and needle sticks.

  • Cannulation should be considered when repeated samples are required, especially over relatively short time periods.
  • In some species, it may be necessary to restrain the animal in some way to stop it removing the cannula. For example, rats are often restrained by a harness, swivel and tether system, which restricts normal movement. Animals should be acclimatised to any restraint system before cannulation.
  • Tethered animals are often housed singly, thus adding to the stress and severity of the procedure. When dealing with social animals, every effort should be made to keep them in social groups. Cannulated pigs, cats and marmosets can be group-housed successfully with appropriate bandaging and protection for the cannula.
  • Cannulation has the potential to cause discomfort to the animal and therefore warrants post-operative administration of analgesics and careful post-operative care and monitoring for the duration of time the cannula is in place.
  • Cannula associated infections can be avoided through the use of sterile equipment and solutions, and by employing an aseptic technique.
  • When selecting any animal for cannulation they should be assessed on health status as well as behavioural temperament. This will help ensure that the animal can cope with the additional handling catheter maintenance and dosing procedures required. 

Please see our dedicated resource on vascular catheters.   

Cardiac puncture

  • Cardiac puncture should only be carried out under deep terminal anaesthesia or on euthanised animals.
  • It is common to exsanguinate animals under terminal anaesthesia using this method. Where the procedure is intended as terminal, death after exsanguination should be ensured via anaesthetic overdose or by incising the heart or major blood vessels.

Volume of blood to be removed

The volume of blood removed and the frequency of sampling should be based on the purpose of the scientific procedure and the total blood volume of the animal. It is essential to take account of the combined effect of sample volume and the frequency of sampling. If too much blood is withdrawn too rapidly, or too frequently without replacement, an animal can go into short-term hypovolaemic shock and/or in the longer term suffer anaemia (see below). Data interpretation and scientific validity may be confounded if excessive sampling is employed.

  • As a general principle, sample volumes and number of samples should be kept to a minimum.
  • As a general guide, up to 10% of the total blood volume can be taken on a single occasion from a normal, healthy animal on an adequate plane of nutrition with minimal adverse effects; this volume may be repeated after three to four weeks. For repeat bleeds at shorter intervals, a maximum of 1.0% of an animal's total blood volume can be removed every 24 hours; the effects of stress, site chosen and anaesthetic used, should be taken into account.
  • If frequent samples are necessary, the use of cannulation as a less stressful alternative to repeated venepuncture should be considered. 

As a general rule, total blood volume can generally be estimated as 55 - 70 ml/kg body weight. However, care should be taken in these calculations as the percentage of total blood will be lower (-15%) in obese and older animals.

Below is a comprehensive table of total blood volumes and safe blood sample volumes for laboratory animals, domestic species and non-human primates (adapted from Wolfensohn & Lloyd 2003, Handbook of Laboratory Animal Management and Welfare, 3rd Edition):

Species

Reference weight (g)

Blood volume (ml/kg)$

Total blood volume (TBV), normal adult (ml)

Safe volume for single bleed (ml)*

Bleed out volume (ml)    

Mouse

18 - 40

58.5

Male 1.5 - 2.4

Female 1.0 - 2.4

0.1 - 0.2

Male 0.8 - 1.4

Female 0.6 - 1.4

Rat

250 - 500

54 - 70

Male 29 - 33

Female 16 - 19

Male 2.9 - 3.3

Female 1.6 - 1.9

Male 13 - 15

Female 7.5 - 9

Hamster

85-150

78

Male 6.3 - 9.7

Female 7.1 - 11.2

Male 0.6 - 0.9

Female 0.7 - 1.1

Male 2.9 - 4.5

Female 3.3 - 5.2

Gerbil

55 - 100

66 - 78

Male 4.5 - 7

Female 3.8 - 6

Male 0.4 - 0.7

Female 0.4 - 0.6

Male 2.2 - 3.5

Female 1.9 - 2.9

Guinea pig

700 - 1200

69 - 75

Male 59 - 84

Female 48 - 63

Male 6 - 8

Female 5 - 6

Male 29 - 42

Female 24 - 31

Rabbit

1000 - 6000

57 - 65

58.5 - 585

5 - 50

31 - 310

Ferret

600- 2000

70

42 - 140

4 - 14

21 - 70

Dog

-

70 - 110#

900 - 1170a

90 - 110

-

Cat

-

47 - 65

140 - 200

14 - 20

-

Pig - Large white

-

56 - 69

13,200 - 15,000

1320 - 1500

-

Pig - Yucatan

-

56-69

4200 - 4800

420 - 480

-

Sheep

-

58 - 64

4060 - 4480

400 - 450

-

Goat

-

57 - 90

3990 - 6300

400 - 630

-

Cattle

-

60

27,000 - 36,000b

2700 - 3600

-

Horse

-

75

33,750 - 45,000b

3375 - 4500

-

Marmoset

-

60 - 70

21 - 24.5

2.1 - 2.4

-

Rhesus macaque

-

55 - 80

Male 420 - 770

Female 280 - 630

Male 42 - 77

Female 28 - 63

-

Long-tailed macaque

-

50 - 96

Male 280 - 560

Female 140 - 420

Male 28 - 56

Female 14 - 42

-

$ A blood volume estimate for a single species may not reflect differences among individual breeds or variations due to age, size, or illness
* Single bleed of 10% total blood volume
# Much breed variation
a Beagle
b assumes adult weight 450-600 kg

The EFPIA/ECVAM good practice guide to the administration of substances and removal of blood (Diehl et al. 2001) also contains recommended mean total blood volumes and maximum blood sample volumes for species of a given bodyweight.

The following decision trees can assist you in determining the amount of blood that can be sampled from mice and rats, and depending on that volume, the most appropriate technique to use. 

Signs of shock and anaemia

It is essential to be able to recognise the clinical signs of shock and anaemia and be able to take appropriate action.

  • Signs of hypovolaemic shock include a fast and thready pulse, pale dry mucous membranes, cold skin and extremities, restlessness, hyperventilation, and a sub-normal body temperature. The Named Veterinary Surgeon should be consulted immediately if shock occurs. If more than 10% of the total blood volume has been removed, a routine replacement with the same volume of warm (30-39oC) normal buffered saline constitutes good animal care.
  • Signs of anaemia include pale mucous membranes of the conjunctiva or inside the mouth, pale tongue, gums, ears or footpads (non-pigmented animals), intolerance of exercise and, at the more extreme level, an increased respiratory rate when at rest. Where there is concern about the development of anaemia, packed cell volume, haemoglobin level, red blood cell and reticulocyte counts should be monitored throughout the series of bleeds using the results from the first sample from each animal as the baseline for the animal.

Resources and references