Blood sampling experiments on laboratory animals
Blood sampling experiments on laboratory animals
Blood collection from laboratory animals is mainly used for routine tests or biochemical and chemical analyses. The choice of blood collection method depends on the amount of blood required for the purpose of the experiment and the type of animal. When the amount of blood used is small, such as red and white blood cell counts, hemoglobin determination, blood smears, and enzyme activity microanalysis, etc., the blood can be collected from capillaries by puncturing the tissues. When a large amount of blood is required, blood can be collected from a vein. When blood is collected from a vein, it should be repeated several times, starting at the end away from the heart to avoid embolization of the entire vein. For example, arterial blood must be taken for the study of the effects of poisons on lung function, blood acid-base balance, and disorders of water and salt metabolism, which require comparisons of arterial and arterial oxygen partial pressures, carbon dioxide partial pressures, and blood pH, as well as the concentrations of K, Na, and CI- ions.
Operation method
Laboratory animal blood sampling method
Principle
There are many methods of blood collection in experimental animals, which can be divided into different parts of the blood collection: tail blood collection (cutting and clipping of the rat's tail), rat tail blood pricking, orbital venous plexus blood collection, severed head blood collection, heart blood collection, jugular vein (arterial) blood collection, abdominal aorta blood collection, femoral arterial (static) vein blood collection, blood collection by clipping of the ear margins, ear vein collection, blood collection of the posterior limb external small saphenous vein, blood collection of the forelimbs inner subcutaneous cephalic veins, blood collection of the capillaries, and blood collection under the wings. Subcutaneous blood collection, capillary blood collection, wing blood collection, etc.
Materials and Instruments
Rabbit, dog, guinea pig. Move I. Blood Collection Methods for Rats and Mice 1. Tail-cutting for blood collection: This method is used when the amount of blood required is small. The animal is first fixed or anesthetized, and the tail is exposed. The tail is immersed in warm water at about 45℃ for a few minutes, or the tail is placed in front of an electric heater and baked for a few minutes, or the tail is rubbed with alcohol to make the blood vessels of the tail fill up, and then the tail is dried up, and the tip of the tail is cut off with a sharp instrument (knife or scissors) to remove the blood from the tail tip of 1--2mm (for mouse) or 3--5mm (for rat). Then the tail was dried, and the tip of the tail was cut off with a sharp instrument (knife or scissors) by 1--2mm (mice) or 3--5mm (rats), and the blood was allowed to flow freely down the wall of the tube or was sucked up with a hemoglobin pipette. At the end of blood collection, the wound was disinfected and pressure was applied to stop bleeding, or fire cauterization (electrical cauterization) was used to stop bleeding or 6% fire cotton glue was applied to stop bleeding. A transverse incision can also be made in the tail, cutting the tail artery or vein, collecting blood and stopping the bleeding in the same way as above. This method of blood collection can generally be collected more than 10 times per rat, each time the mouse can be collected about 0.1 ml of blood, the rat can be collected 0.3-0.5 ml of blood, such as the appropriate method, the room temperature is higher, the mouse can also be collected more than 0.5 ml of blood, the rat can be collected more than 3 ml of blood. 2. Rat tail prick method: When the amount of blood collected from rats is very small (for white blood cell count, hemoglobin check or blood sugar test), this method can be used. The rat's tail is first wiped with warm water, then sterilized and wiped with alcohol to make the rat's tail congested with blood, and then punctured into the rat's tail vein with a No. 7 or No. 8 needle, and blood drips out when the needle is withdrawn. If the blood is collected repeatedly for a long period of time, it should be punctured close to the end of the rat's tail first, and then punctured towards the proximal end gradually. 3. Blood collection from the retro-orbital vein plexus: the left hand grasps the skin of the head between the two ears of the rat, fixes the head, and gently presses down the sides of the neck, causing difficulties in the venous blood return of the head, so that the eyeballs protrude outwardly, and the retro-orbital vein plexus is filled with blood. The right hand holds a glass blood collection tube (capillary inner diameter of 1 - 1.5 mm, capillary segment length of about 1 cm, the other end of the gradual expansion into a trumpet) of 7 - 10 cm in length or a 1 ml syringe connected to a 7-gauge needle, so that the blood collection device and the surface of the rat make a 45°C The needle was inserted between the lower eyelid and the eyeball and gently moved towards the base of the eye, where the blood collection tube was rotated to incise the venous plexus. The blood collection tube is held in a horizontal position and the blood is removed with a slight suction. Once the desired amount of blood is obtained, the pressure on the neck is removed and the blood collector is pulled out to prevent postoperative bleeding from the puncture hole. Generally, the depth of puncture is about 2--3mm in mice and 4--5mm in rats. When puncturing, stop advancing when resistance is felt, and at the same time, withdraw the needle about 0.1--0.5mm, which can be withdrawn while withdrawing. At the same time, withdraw the needle about 0.1 - 0.5mm, and extract blood while withdrawing. Note: Mice, rats, guinea pigs, rabbits can collect blood from the retro-orbital venous plexus, according to the experimental needs, can be repeated in a few minutes in the same puncture hole blood collection, generally the two eyes rotating blood collection, mice each time to collect blood 0.2-0.3 ml, rats each time to collect 0.5-1.0 ml of blood. The blood can be collected from 0.2-0.3 ml in mice and 0.5-1.0 ml in rats. 4. Blood collection from the heart: The heart of rodents is smaller and has a faster heart rate, so blood collection from the heart is more difficult and is generally less frequently used, and mice hardly use blood collection from the heart in vivo. When collecting blood from rats, the rats will be fixed on the fixed board in supine position, the hair of the precordial area will be cut off, and the skin will be sterilized with iodine alcohol, in the left side of the 3rd-4th intercostal space, the left index finger will touch the place of the heart beat, and the right hand will take the syringe connected with a 4-5 needle, and choose the place where the heart beat is strongest to puncture. Choose the strongest heart beat to puncture, when the needle into the heart, the blood from the heart beating force automatically into the syringe, can also be held by the left hand rats, the right hand to choose the strongest heart beat directly after the needle into the heart cavity, the heart of the blood collection is best to stab the heart, repeated stabbing of the heart, it will cause the death of the animal. It is also possible to open the chest to collect blood in one death, first the animal for deep anesthesia, open the chest cavity, expose the heart, use the needle to pierce the right ventricle, suction blood collection. 5. Orbital artery or orbital vein blood collection: first, the animal will be fixed with the left hand upside down, pressure on the neck (back), so that the eyeballs protruding and congested, with a small curved hemostat (or forceps) to quickly clamp the eyeballs (or gouge the eyeballs), and the head of the forceps to poke through the posterior periosteum of the eyeballs, the orbital quickly outflow or spray blood, blood drops into the pre-inflated with anti-coagulant vessels, until the required amount of blood collection, generally take the animal body weight of 4-5% blood volume. Generally, 4%-5% of the animal's body weight can be taken as blood volume. If the blood in the eye coagulates during blood collection, the opposite eyeball can be gouged out again, and the chest cavity can be squeezed regularly and loosely to induce the blood to flow out. This method of blood collection is a one-time blood collection, and the animals mostly die after blood collection. 6. Jugular vein or carotid artery blood collection: Anesthetize the animal and fix it on the back, cut off the hair on the outside of one side of the neck, dissect the neck and separate and expose the jugular vein or carotid artery, and then pierce it along the jugular vein or carotid artery in a parallel direction with the syringe needle to extract the required amount of blood, which is about 0.6 ml in mice and about 8 ml in rats. The jugular vein or carotid artery can also be cut, and the blood flowing out can be sucked up with a syringe (without a needle), or blood can be collected with a test tube. 7. Blood collection from femoral vein or femoral artery: After anesthesia and dorsal fixation, cut the skin of the left or right groin, separate the femoral vein or femoral artery, pierce the injection needle into the vein or artery parallel to the blood vessel, and then pull the needle slowly to collect blood. Can also not be anesthetized and not surgical animals, first by the assistant to hold the animal, the blood collector's left hand to straighten the animal's lower limbs, so that the veins are full, or to beat as an indicator, the right hand with a syringe into the blood vessels to collect blood. Generally, mice can collect about 0.2 - 0.8 ml of blood, rats can collect about 0.4 - 1.6 ml of blood, such as continuous multiple femoral vein blood collection, the blood collection site should be selected as close as possible to the centrifugal end. 8. Abdominal aorta blood collection: after anesthesia, the animal was fixed in supine position, and the abdominal cavity was cut from the midline of the abdomen, so that the abdominal aorta was exposed, and the blood was collected with a syringe: the distal end of the abdominal aorta could also be ligated, the proximal end of the abdominal aorta could be blocked by a blocker, and then the proximal end of the abdominal aorta could be pierced parallelly in the middle of the abdominal aorta and the proximal end of the proximal end of the proximal end of the proximal end of the abdominal aorta could be loosened, and the blood was collected immediately. 9. Blood collection by severing the head: the thumb and forefinger of the left hand hold the skin of the rat's neck more tightly from the back and turn the animal's head downward, the right hand cuts the rat's neck fiercely with scissors, cuts about 1/2 - 4/5 of the neck, and lets the blood flow into the container, the blood can be collected from the mice 0.8 - 1.2 ml, rats 5ml, and rats 5ml, and then the blood can be collected from the rat. 1.2 ml for mice and 5-10 ml for rats. care should be taken to prevent animal hair and other debris from flowing into the container and causing hemolysis when collecting blood. Methods of blood collection in guinea pigs 1. Blood collection from the ear margin: after sterilizing the ear, use a sharp instrument (knife or razor blade) to cut the blood vessels at the ear margin, and the blood will flow out from the blood vessels. 0.5ml of blood can be collected by this method, and the blood can be stopped by pressure with sterilized gauze for 5-10 seconds after blood collection. 2. Blood collection from the heart: fix the guinea pig in supine position on a fixed board, cut off the hair in the precordial area and sterilize the skin with iodine alcohol, touch the heart beat with the index finger of the left hand at the left 4-6th intercostal space, and take a syringe with a 4-5 needle attached to it with the right hand. The right hand takes a syringe with a 4-5 gauge needle attached to it and selects the place where the heart beat is strongest to puncture. When the needle is punctured into the heart, the blood automatically enters the syringe by the force of the heart beat. 3. Blood collection from the dorsal mid-foot vein: the assistant fixes the animal and brings it to the operator with its right or left hind knee straightened. The operator sterilizes the dorsum of the animal's foot with alcohol, finds the dorsal mid-foot vein, pulls the toe end of the guinea pig with the thumb and forefinger of the left hand, and stabs the vein with a syringe needle in the right hand. Bleeding was done immediately after the needle was withdrawn, and blood was aspirated with a pipette for experimental use. After blood collection, the bleeding was stopped by pressure with gauze or skimmed cotton. When repeated blood collection, the two hind limbs should be alternated. In addition, guinea pigs can also use the femoral artery blood collection, orbital venous plexus blood collection, jugular vein blood collection, etc., with reference to the method of blood collection in mice. Blood collection methods for rabbits 1. Blood collection from the heart: the rabbit should be fixed in supine position, and the left hand should be used to touch the heart beat, and choose the most obvious place of heart beat for puncture. The puncture site is the 3rd rib space, the right edge of the sternum at 3mm, each time the blood collection is not more than 20 - 25ml, the application of this method can be injected into the heart cavity and blood collection, generally after 6 - 7 days, can be repeated for cardiac puncture. 2. Ear margin vein blood collection: after fixing the animal, expose both ears, choose the ear with clear vein to remove hair, routinely disinfect, press the root of the ear to make the vein full, then you can use the needle to puncture the vein to collect blood. One time blood collection can be about 5ml. Note: If a small amount of blood is taken for general routine examination, it can be waited until the veins at the edge of the ear are filled with blood, and then the blood vessels near the tip of the ear can be punctured with a No. 5 1/2 needle, and the blood will flow out of the punctured opening. 3. Blood collection from the central artery of the rabbit's ear: place the rabbit in the rabbit fixation box, in the center of the rabbit's ear, there is a thicker, more brightly colored central artery, fix the rabbit's ear with the left hand, and with the right hand, take the syringe, and at the end of the central artery, pierce the artery in a parallel way along the artery in a centripetal direction, which means that the arterial blood can be seen to enter the syringe, and the blood can be collected with the use of medicated cotton to stop the hemorrhage by pressure. This method can be up to 15 ml of blood at a time. the needle used for blood collection is usually a No. 6 needle, not too fine, the needle should start from the end of the central artery, do not collect blood near the root of the ear, because the root of the ear is thick soft tissues, blood vessels are slightly deeper, and it is easy to penetrate the blood vessels, resulting in subcutaneous hemorrhage. The central artery of the rabbit's ear is prone to spasmodic contraction, therefore, before drawing blood, the rabbit's ear must be fully engorged with blood, when the artery is dilated, before spasmodic contraction occurs, the blood must be drawn immediately. 4. Blood collection from subcutaneous veins in the tibia of the hind limb: after fixing the rabbit in supine position (or fixing the rabbit by one person), pulling off the hair of the tibia, and tying a rubber tube at the upper end of the tibia, subcutaneous veins can be seen clearly under the superficial skin of the outer side of the tibia. The vein was fixed with two fingers of the left hand, and a syringe with a No. 5 1/2 needle was inserted into the vein in a parallel direction with the right hand, and blood was collected when the needle was pumped into the syringe. The blood can be collected in 2-5 ml at a time, and after blood collection, a cotton ball is used to press the site of blood collection to stop bleeding, which usually takes 1 minute. If the bleeding is not stopped properly, it can cause subcutaneous hematoma, which will affect the blood collection for many times. 5. Blood collection from femoral vein: After the animal is anesthetized and fixed in supine position for femoral vein exposure and separation surgery, when blood is collected from femoral vein, the syringe is parallel to the blood vessel, and it is inserted in the direction of heart from the lower end of the femoral vein, and then blood can be collected by twitching the needle plug. The amount of blood collected is large, usually more than 10 ml can be taken at one time. After blood collection, use dry gauze or cotton ball to gently press the site of blood collection to stop bleeding. 6. Jugular vein blood collection: After the animal is fixed in supine position under anesthesia, the jugular vein is exposed and separated, and the syringe is inserted in the parallel direction of blood vessels from the proximal end of the vein (2-3cm from the vein branch) to the cephalic end, so that the syringe needle is drawn to the bifurcation of the main jugular vein branch, and then the blood can be collected, and the blood vessels here are thicker, and the volume of blood can be collected in larger quantities, and more than 10 ml can be collected in general. Here the blood vessel is thicker and the volume of blood collected is larger, usually more than 10ml can be taken at one time. After blood collection, dry gauze should be used to gently press the blood collection site to stop bleeding. Methods of blood collection for dogs and cats 1. Blood collection from the lateral saphenous vein of the hind limb and the subcutaneous cephalic vein of the forelimb: this method is most commonly used. The lateral small saphenous vein of the hind limb is superficial and subcutaneous in the lower 1/3 of the tibia of the hind limb. When drawing blood, the dog will be fixed on the dog frame or make the dog lying on its side, the dog will be fixed by the assistant, the hair of the blood drawing area will be clipped, iodine-alcohol disinfection, the blood collector will use the left thumb and forefinger to hold tightly to the upper part of the clipped area, so as to make the veins of the lower limbs filled, the right hand will use the sterilized syringe connected with the No. 6 or No. 7 needle to quickly penetrate into the vein, and the left hand will relax to fix the needle, and the blood will be drawn at an appropriate speed (no bubbles is preferred). Alternatively, a rubber band can be tied around the dog's femur or an assistant can hold the femur tightly. If only a small amount of blood is needed, simply pierce the vein directly with the needle and wait for the blood to drip naturally out of the needle hole into a receptacle or make a smear. When collecting blood from the cephalic vein under the skin of the inner forelimb, the procedure is the same as above. Generally, a dog can collect 10-20ml of blood at one time. 2. 2. Blood collection from the vein at the edge of the ear: this method is suitable for experiments that require a small amount of blood. Trained dogs generally do not need to tie the mouth, cut off the tip of the ear short hairs, that is, the visible ear marginal vein, with 75% alcohol disinfection of the local, dry, gently rubbing the ear with a finger, so that the vein is dilated, with a syringe with 5 1/2 needle in the ear at the end of the marginal vein puncture the blood vessels, blood to be leaked out of the collection of blood, or will be against the direction of the blood flow of the needle pricked ear marginal vein collection of blood, blood collection, after the use of cotton wool to stop the pressure of hemorrhage. 3. Jugular vein blood collection: the dog does not need anesthesia, trained dogs do not need to be fixed, untrained dogs should be fixed, take the lateral position, cut off the neck hair, iodine, alcohol disinfection of the skin, straighten the dog's neck, tilt the head as far as possible back, the left thumb pressed the vein injected into the chest position of the skin, so that the jugular veins are anguished, right hand take the syringe with a 6 1/2 needle, the needle along the blood vessels in the parallel direction of proximal to the end of the blood vessels. The left hand fixes the needle, and more blood can be obtained at one time. However, this vein is easy to slide under the skin, the needle should be accurate when stabbing, and the needle should be fixed with the hand after stabbing. Anesthesia is needed to anesthetize the dog when collecting blood from an irritable dog. 4. Femoral artery blood collection: This method is commonly used for canine arterial blood collection. Trained dogs, in the awake state of the dog lying position fixed on the canine anatomy table, stretch the hind limbs straight outward, exposure of the inguinal deltoid artery pulsation site, cut off the hair, iodine-alcohol disinfection, the left hand middle finger, forefinger probe the femoral artery pulsation site, and fixed the blood vessels, the right hand take the syringe with a No. 5 1/2 needle, the needle by the arterial pulsation into the blood vessels, such as blood, can be slightly rotated needle or If no blood is seen, the needle can be turned slightly or moved up and down slightly, and the blood can enter the syringe automatically. If the blood is not seen, the needle can be turned slightly or moved up and down, and the blood can enter the syringe automatically. If the blood is inserted into the vein, it must be withdrawn and re-punctured, and after drawing blood, the needle should be withdrawn quickly, and the local area should be pressed with a dry cotton pad for 2-3 minutes to stop the bleeding. 5. Blood collection from the heart: after anesthesia, the dog was fixed on the operating table, the forelimbs were fixed in the dorsal direction, the chest was exposed, the hair on the left side of the 3rd-5th intercostal space was clipped, the skin was sterilized by iodine and alcohol, and the collector of blood touched the left side of the 3rd-5th intercostal space with the left hand, and chose the place where the heartbeat was most obvious to enter the needle. The left hand of the blood collector should touch the left 3-5 intercostal space and choose the place where the heartbeat is the most obvious to enter the needle, generally at the 4th intercostal space 1cm outside the left edge of the sternum, take the syringe with a 6 1/2 needle (or 7 needle), and vertically pierce into the heart toward the dorsal side of the animal, and the blood collector can feel the heartbeat along with the needle contact and adjust the direction and depth of the pierce, and the angle of the oscillations should be as small as possible, so as to avoid damaging the myocardium too much, or cause hemorrhage in the chest cavity. When the needle correctly pierces the heart, the blood can automatically enter the syringe, and this method can extract a large amount of blood. Note: The blood collection method of cats is basically the same as that of dogs. Commonly used methods are subcutaneous cephalic vein of the forelimbs, femoral vein of the hind limbs, blood collection in the vein at the edge of the ear, and a large number of blood samples can be taken from the jugular vein or the heart, with the same method as the previous one. Monkey's blood collection methods Monkey's blood collection methods are similar to that of human beings. 1. Capillary blood collection: after local disinfection, the monkey's thumb or heel is punctured to collect blood. 2. Venous blood collection: when a large amount of blood is needed, venous blood collection is used, and the most suitable parts are the subcutaneous veins of the hind limbs and the external jugular vein. The most suitable sites are the subcutaneous veins of the hind limbs and the external jugular vein. Fix the monkey on the monkey table, lying on its side, with its head slightly lower than the table surface, the assistant fixes the monkey's head and shoulders, cuts off the neck hair first, after iodine-alcohol disinfection, presses the left thumb to hold the external jugular vein which is located in the middle point between the angle of the maxilla and the clavicle in anger, and then the right hand holds a syringe with a No. 6 1/2 needle to collect blood in the same way as the human venous collection method, and after blood collection, the medicinal cotton is pressed locally to stop the hemorrhage. Blood can also be collected in the elbow socket, carpal bones, the dorsum of the hand and the dorsum of the foot from selected veins, but these parts of the veins are thin, easy to slide, difficult to puncture, slower blood flow, and slightly difficult to collect blood. Blood collection from subcutaneous veins of the hind limbs is similar to that of dogs. Common Problems How to avoid hemolysis in blood specimens, details to be noted: 1. Laboratory animals blood collection needle inner wall is not smooth Classification of blood collection needles: (1) Stainless steel needles: the outer wall is smooth, the inner wall seems smooth, the substance is rough, easy to scratch the cells, causing hemolytic cascade reaction, resulting in hemolysis of blood specimens. (2) Intravenous retention needles: smooth outer wall, smooth inner wall, can avoid scratching cells, effectively preventing hemolysis caused by the blood collection process, but the cost is higher. 2、Blood specimen containers are not clean. 3、Laboratory animals mixed with animal hair The operation of blood collection process is not competent and clean enough, which causes animal hair and other foreign matter to mix or contact with the blood specimen, causing hemolysis. 4、Laboratory animals back to pump too fast, too much pressure In the effective cross-section of the needle back to the pumping speed, the greater the pressure, the cells are subjected to extrusion, which can easily lead to rupture, causing hemolysis. 5、Laboratory animals blood from the syringe into the container without removing the needle When blood is injected into the vacuum blood collection vessel, the vacuum pressure is too high, causing cells to be squeezed sharply and ruptured, causing hemolysis. The needle should be removed and the blood should be injected slowly along the wall of the tube without bubbles. 6、The formation of air embolism during blood collection in experimental animals. Blood collection can only be pumped outward, not pushed into the blood vessel, so as not to inject air to form air embolism, resulting in hemolysis. 7. Factors of experimental animals Hemolysis caused by experimental factors, such as the drug chloral hydrate, itself has a slight hemolytic effect. 8、Vigorous shock during transportation of experimental animals Avoid hemolytic reaction caused by violent shock during the transportation of blood specimens. For more product details, please visit Aladdin Scientific website.
Xylene Alcohol Liquid paraffin
Vascular clamps Needles Blades Cotton balls Rabbit immobilizer Syringe Surgical table Scissors Gauze
9, Source China Laboratory Animal Information Network