Collection of blood specimens

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Collection of blood specimens

Two different kind of blood specimens can be collected for infectious disease diagnosis (which makes exception of blood count and biochemistry):

  • the whole blood for pathogen identification,
  • the serum for serological diagnosis.

Blood samples are collected by venal puncture on live animal, as cleanly as possible, using sterile equipment, and with respect for the welfare of the animal:

  • Use new needle and syringe for each animal,
  • Dispose needles properly,
  • Clean the puncture area with 70% alcohol,
  • Handle animals calmly and using appropriate restraint,
  • Although most techniques do not require large amount of blood it is preferable to collect at least 2.5mL of blood, but ideally 10mL in large animals, and 4-6mL in small animals,
  • Identify the sample with a permanent method and fill the sampling form at the time of sampling.

Where the samples is taken from depends on the type of animal and fields conditions. When blood is taken from small veins it is better to use a syringe instead of a vacutainer to prevent the vein collapsing. Possible sites include:

  • Jugular veins (large animals),
  • Caudal vein (cattle),
  • Brachial, brachiocephalic, femoral, tarsal veins,
  • Cranial Vena cava in pigs,
  • Mammary veins,
  • Ear veins in pigs: 20 gauge needle,
  • Saphenous veins, retro-orbital veins, intracardial punction in rodents, small vertebrates, use a 23 gauge needle,
  • Wings (brachial) in chicken, use 20 to 23 gauge needle.

Processing and conservation of samples depend on the type of examination to be undertaken. 

Whole blood is prone to hemolysis, which is caused by red cells and interferes with a number of diagnostic tests. To help prevent hemolysis:

  • Preferentially use a vacutainer instead of a syringe, if you use a syringe fill it with negative pressure,
  • If you have to transfer the blood from a syringe to a sample container, remove the needle first and transfer slowly and smoothly,
  • Do not shake the blood,
  • Do not freeze whole blood.

Whole blood samples

For many infectious diseases, the agent can be identified from blood, by detection of antigens or culture and isolation of the agent, or demonstration of DNA.

In order to do such examinations the blood needs to be stored properly , with an anticoagulant. Different anticoagulants are available in commercial tubes such as: EDTA (purple tubes) or Heparin (green tubes) which are commonly used for bacteriology or virology, they have specific proprieties and it is better to refer to disease factsheet and/or laboratory to know which is the best anticoagulant to use. However EDTA is often recommended.

Blood is carefully transfer from the vacutainer or the syringe to the tube and gently invert 2-3 times to thoroughly mix the anticoagulant with the blood.

For virology antibiotics may be added, it is better to contact the laboratory first to have technical recommendations.. The usual method is to add penicillin and streptomycin to give final concentrations of 200 units of penicillin and 200 mg of streptomycin per ml of blood.

The blood container should be firmly capped and taped to prevent leakage. 

Whole blood samples should be sent to the laboratory in an insulated container with ice at 4°C. They should not be frozen. Blood samples for virology must be sent to the laboratory within 24 to 72 hours depending on virus survivability.

Blood for serum samples

Serum samples are used for serological analysis. A positive serological reaction does not necessary means that the animal is clinically infected: vaccination, post-infection immunity, persistence of maternal antibodies in young animals, and lack of specificity of the test, are common cause of "false" positive. 

Therefore, when possible it is always better to provide either paired samples from the same animal with 2-3 weeks between sampling or from different animals at different stage of the disease.

However in an outbreak of an exotic disease, a single positive reaction should be considered highly significant.

To collect serum from the blood it is important to let the tubes stand at ambient temperature for 1-2 hours in an upright position to let the clot begin to contract. The clot can be removed using a sterile rod after tubes are placed at 4°C for 12 hours. The serum can then be removed with a pipette or decanted in fresh tubes but if necessary the sample can centrifuged at 1-3000g for 10 minutes and the clear serum removed . It is possible to freeze serum at -20°C or even colder to preserve samples for later analysis. Plasma samples can also be used for serological diagnosis (blood should be cooled in an ice bath and centrifuged as soon as possible; then the plasma should be separated immediately after centrifuging).

If a bacterial disease is suspected, it is often useful to keep and submit the clot to the laboratory for bacterial culture.

Hemolysis can interfere with many serological diagnosis tests.