Blood collection and plasma separation

Blood Collection

For the purpose of conducting serological, immunological, and hematological tests, it is necessary to collect blood from the animal during the blood test. Given the circulatory system in rodents and its high similarity to that of humans, valuable information can be obtained. Blood circulates throughout the body and is in contact with nearly all cells. Therefore, changes in various parts of the animal's body affect the blood, and most of these changes are traceable. Various methods exist for blood collection from animals. Some methods lead to the collection of the entire blood volume, ultimately resulting in the animal's death. Thus, the choice of blood collection method depends on the required blood volume and whether the animal needs to remain alive for further testing. Blood can be collected from various sites such as the tail, saphenous vein, eyes, heart, etc. It should be noted that blood collection from a conscious animal can increase stress and lead to alterations in the animal's biochemical factors. Therefore, for more accurate and reliable results, it is preferable to anesthetize the animal before proceeding with blood collection. Generally, the blood volume in rodents is about 10% of their body weight or approximately 55 to 80 milliliters per kilogram of body weight. However, this amount may decrease by up to 15% in older animals compared to younger ones. The maximum amount of blood that can be extracted from rodents is between 6% to 8%, and removing this amount will result in the animal's death. Additionally, the maximum volume of blood that can be safely collected without harming the animal is about 1% of its total body weight. The interval required for re-blood collection is two weeks.

In laboratory animal blood collection, sudden removal of the syringe needle from the heart or vein should be avoided, as this can lead to hemolysis of red blood cells and the release of hemoglobin and non-separable coloring compounds into the blood. Furthermore, when transferring blood from the syringe to the tube, the needle tip must be removed. To obtain serum, blood is collected in a tube devoid of any anticoagulant and is then centrifuged after clotting. For plasma extraction, blood is exposed to anticoagulants such as EDTA or heparin and then centrifuged. Serum lacks clotting factors such as fibrinogen because, during serum preparation, the blood clots and the clotting factors precipitate. However, in plasma separation, clotting factors are present.

General Principles of Blood Collection in Animals

The blood collection method must be described in the approved protocol by the animal ethics committee. These methods should be minimally painful and stressful. Blood samples may be collected under anesthesia or without it. Adequate training for blood collection using any method in any species is essential. Generally, blood samples are withdrawn from venous, arterial, or cardiac blood vessels.

The frequency of blood collection is important. Once every two weeks is ideal for non-rodents. This study may require several blood samples, utilizing lagomorphs (e.g., rabbits). The total indirect blood collection without fluid replacement is limited to 10% of the total circulating blood volume in healthy, normal adult animals, and the collection may be repeated after 3 to 4 weeks. If repeated blood samples are required at short intervals, a maximum of 0.6 milliliters per kilogram per day or 1.0% of the total blood volume in animals may be removed every 24 hours.

If the study involves repeated blood sampling, samples can be obtained through a temporary cannula. This may reduce pain and stress in experimental animals. The estimated blood volume in adult animals ranges from 55 to 70 milliliters per kilogram of body weight. Caution should be exercised in older and obese animals. If the volume of blood collected exceeds 10% of the total blood volume, fluid replacement may be necessary. Lactated Ringer's solution (LRS) is recommended by the National Institutes of Health (NIH) as the best fluid replacement. If the volume of blood collected exceeds 30% of the total circulating blood volume, adequate care must be taken to prevent the animal from becoming hypovolemic.