采集实验动物血液 z-bo 2020-10-13 340

　　In experimental research, it is often necessary to collect the blood of experimental animals for routine inspection or certain biochemical analysis, so it is necessary to master the correct collection, separation and storage of blood. The choice of blood sampling method is mainly determined by the amount of blood required for the purpose of the experiment and the type of animal. Any test with less blood volume, such as red and white blood cell counts, hemoglobin determination, blood smears, and enzyme activity microanalysis methods, can puncture tissues and take blood from capillaries. Venous blood can be collected when the blood volume is large. If you need to repeat venous blood collection many times, you should start away from the heart to avoid embolism and affect the entire vein. For example, to study the effects of toxicants on lung function, blood acid-base balance, and water-salt metabolism disorders, it is necessary to compare arterial and venous blood oxygen partial pressure, carbon dioxide partial pressure, blood pH, and K+, Na+, and Cl- ion concentrations. Arterial blood must be used. Attention should be paid to blood collection: ①There is sufficient light in the blood collection area; the room temperature should be kept at 25～28℃ in summer and 15～20℃ in winter; ②blood collection equipment and parts generally need to be disinfected; ③syringes and syringes for blood collection The test tube must be kept clean and dry; ④If anticoagulant whole blood is required, an anticoagulant must be added to the syringe or test tube in advance. Now the blood collection methods are introduced separately by animal and part.

　　(1) Blood sampling method for mice and rats

　　1. Cut (cut) the tail to collect blood. This method is used when the amount of blood required is small. Fix the animal and expose the tail. Cut off the tail hair and sterilize it, then soak it in warm water at about 45°C for a few minutes to fill the tail blood vessels. Dry the tail again, cut off the tip of the tail 0.3-0.5cm with a sharp instrument (knife or scissors), let the blood drip freely into the container or suck it with a hemoglobin pipette. After the blood collection is completed, the wound is disinfected and the bleeding is suppressed. You can also make a transverse incision in the tail to cut the tail artery or vein, and the method of collecting blood is the same as above. Each mouse can generally collect blood more than 10 times. Each time 0.1ml of blood can be taken for mice and 0.3～0.5ml for rats.

　　2. Rat tail pricking blood method This method can be used when the blood volume of rats is not much (only for white blood cell count or hemoglobin check). First wipe the tail of the rat with warm water, then disinfect and wipe with ethanol to make the tail congested. Use a No. 7 or No. 8 injection needle to pierce the tail vein of the rat. When the needle is pulled out, blood will drip out, and 10-50mm3 can be collected at a time. If the blood is taken repeatedly for a long time, the puncture should be close to the end of the tail of the rat first, and then gradually puncture to the proximal end.

　　3. Blood sampling from the orbital venous plexus The left thumb and showing two fingers of the blood sampling person should hold the mouse or rat neck tightly from the back (rats need to wear gauze gloves for blood sampling) to prevent the animal from suffocating. When blood was taken, the left thumb and index finger gently pressed both sides of the animal's neck to congest the retroorbital venous plexus. Continue to connect a 1ml syringe with a 7 gauge needle or a long-necked (3～4cm) hard glass dropper (capillary inner diameter 0.5～1.0mm) in the right hand, make the blood collection device and the mouse surface at an angle of 450, pierce through the inner corner of the eye, and the needle The bevel faces the eyeball first, and then turn around 1800 to make the bevel face the posterior boundary of the eye socket. The penetration depth is 2～3mm for mice and 4～5mm for rats. When you feel resistance, stop advancing, and at the same time, withdraw the needle by 0.1～0.5mm, and draw it while retracting. If the puncture is proper, blood can flow into the capillary naturally. When the required blood volume is obtained, the pressure on the neck is removed and the blood sampling device is pulled out to prevent postoperative bleeding from the puncture hole.

　　If you are skilled, there is no difficulty in repeating blood sampling in a short period of time with this method. It is better to rotate the left and right eyes. Mice weighing 20-25g can collect blood 0.2-0.3ml each time; rats weighing 200-300g can collect blood 0.5-1.0ml each time, which is suitable for the inspection of certain biochemical items.

　　4. Decapitation to take blood. The left thumb and index finger of the blood collector should hold the neck skin of the big (small) mouse tightly on the back and make the animal head down. Cut the rat's neck with scissors with the right hand, cut off the front of 1/2～4/5 of the neck, and let the blood drip into the container freely. 0.8～1.2ml of blood can be collected for mice and 5～10ml for rats.

　　5. Heart blood collection Rats have a small heart and a faster heart rate. It is difficult to collect blood from the heart, so use less. The method of blood collection in vivo is the same as that of guinea pigs. If the chest is opened and the blood is collected for one death, the animal is first deeply anesthetized, the chest cavity is opened, the heart is exposed, and a needle is inserted into the right ventricle to draw blood. 0.5-0.6ml blood is collected for mice and 0.8-1.2ml for rats.

　　6. Jugular arteriovenous blood collection First, the animal is fixed in the supine position, the neck skin is cut, and the subcutaneous connective tissue is separated, so that the jugular vein is fully exposed, and the blood can be sucked out with a syringe. Separate the carotid arteries on both sides of the trachea, ligate the centrifugal end, and cut the centripetal end to drop the blood into the test tube.

　　7. Blood collection from the abdominal aorta It is best to first anaesthetize the animal, fix it on the surgical frame, and cut the abdominal cavity from the midline skin of the abdomen to expose the abdominal aorta. Aspirate blood with a syringe to prevent hemolysis. Or use toothless forceps to peel off the connective tissue, clamp the proximal end of the artery, cut the artery with pointed surgical scissors, and spray the blood into the container.

　　8. Femoral movement (static) pulse blood sampling First, the assistant holds the animal, and the blood sampling person straightens the animal's lower limbs with his left hand to fill the vein. Or use the pulsation as an indicator and pierce the blood vessel with a syringe with your right hand. The blood is collected from 0.2 to 0.8 ml for mice weighing 15 to 20 g, and 0.4 to 0.6 ml for rats.

　　(2) Guinea pig blood collection method

　　1. Blood collection by cutting the ear edge After disinfecting the ear, cut the ear edge with a sharp instrument (knife or blade) and apply 20% sodium citrate solution on the edge of the incision to prevent blood clotting. The blood can flow out of the incision automatically and enter the container. During operation, the effect of congesting ears is better. This method can collect about 0.5ml of blood.

　　2. Cardiac blood collection Before blood collection, the strongest part of the heartbeat should be identified, usually in the middle of the left edge of the sternum, and the most obvious part of the heartbeat should be selected for puncture. The needle should be slightly slender to avoid bleeding from the puncture hole after the operation. Refer to the rabbit heart blood collection for the operation method. Due to the small body of the guinea pig, it is generally not necessary to fix the animal on the dissection table, but the assistant can hold the fore and hind limbs to collect blood. Adult guinea pigs should not exceed 10ml of blood per week.

　　3. Muscle arterial blood sampling Fix the animal on the operating table in supine position, cut off the hair in the groin area, after anesthesia, disinfect with iodine locally. Cut the skin 2 to 3 cm long to expose and separate the femoral artery. Then, lift the femoral artery with forceps, ligate the distal end, clamp the proximal end with hemostatic forceps, cut a small hole in the center of the artery, insert it with a sterile glass small catheter or polyethylene or PTFE tube, release the hemostatic forceps, The blood flows out of the catheter port. 10-20ml of blood can be collected at one time.

　　4. Take blood from the vein of the dorsal foot. The assistant fixes the animal and lifts its right or left and right knee joints to the front of the surgeon. The surgeon sterilized the back of the animal's foot with ethanol, and found the dorsal midfoot vein, then pulled the toe end of the guinea pig with the thumb and index finger of the left hand, and pierced the vein with the injection needle in the right hand. Bleeding immediately after the needle was removed, showing a hemispherical bulge. After blood collection, pressure with gauze or absorbent cotton to stop bleeding. When blood sampling is repeated, the two hind limbs are used alternately.

　　(three) blood sampling

　　1. Ear vein blood collection This method is one of the most commonly used blood collection methods. It is often used for repeated blood collection. Therefore, it is particularly important to protect the ear veins and prevent embolism. Put the rabbit into a fixed box with only the head and two ears exposed, or be held by an assistant. Select ears with clear ear veins, pluck the hairs from the ear veins, disinfect locally with 75% ethanol, and leave to dry. Gently rub the rabbit ears with your fingers to expand the veins. Use a syringe connected with a 5 (1/2) gauge needle to puncture the blood vessels at the end of the ear vein. When the blood leaks out, take blood or pierce the needle into the ear against the direction of blood flow Intravenous blood collection, after blood collection, use cotton ball compression to stop bleeding, this blood collection method can collect blood at most 5-10ml at a time.

　　2. Blood collection from the central artery of the ear Put the rabbit in the fixed tube of the rabbit. There is a thicker, brighter red central artery in the center of the rabbit ear. Fix the rabbit ear with the left hand and take the syringe with the right hand. At the end of the central artery, parallel to the artery. Penetrate the artery to the heart to see the arterial blood enter the syringe. Pay attention to stop bleeding after the blood is taken. This method can draw blood up to 15ml at a time. However, attention should be paid when drawing blood. Since the central artery of rabbit ears is prone to spastic contraction, the rabbit ears must be fully congested before blood drawing. When the artery is expanded and no spastic contraction occurs, blood should be drawn immediately. If the waiting time is too long , Arteries often have spastic contractions for a long time. The needle used for blood collection is generally a 6-gauge needle, not too thin. The acupuncture site starts from the end of the central artery. Do not take blood near the base of the ear, because the soft tissue at the base of the ear is thick and the blood vessels are slightly deeper, which can easily penetrate the blood vessels and cause subcutaneous bleeding.

　　3. Heart blood collection Place the rabbit on its back and set the injection needle vertically into the heart at 3mm from the left edge of the sternum in the third intercostal space. The blood then enters the needle tube. Precautions are as follows: ①The action should be rapid to shorten the time of needle retention in the heart and prevent blood clotting; ②If the needle has entered the heart but does not bleed, the needle should be moved back slightly; ③The needle should not swing side to side in the chest cavity To prevent injury to the heart and lungs, 20-25ml of blood can be taken at one time.

　　4. Blood is taken from the subcutaneous vein of the hind limbs. The rabbit is fixed on the rabbit fixed plate in the supine position, or the rabbit is fixed by one person. Pull out the coat of the tibia, and tie a rubber tube to the upper thigh of the tibia, and the subcutaneous veins can be clearly seen under the superficial skin on the outer side of the tibia. Use the left hand and two fingers to fix the vein, take a syringe with a 5 (1/2) gauge needle and puncture the blood vessel in parallel to the subcutaneous vein, draw the needle plug, if blood enters the syringe, it means that the needle has penetrated the blood vessel. Blood can be taken. Take 2～5ml at a time. After the blood is taken, a cotton ball must be used to press the blood-taken site to stop the bleeding, which takes a little longer, so it is not easy to stop the bleeding. If the bleeding is not properly stopped, it can cause subcutaneous hematoma and affect the continuous blood sampling.

　　5. Blood collection from femoral vein and jugular vein First, perform the operation of exposure and separation of femoral vein and jugular vein.

　　(1) Femoral vein blood collection: The syringe is parallel to the blood vessel and inserted into the heart from the lower end of the femoral vein. The blood can be taken by pulling the needle slowly. Pay attention to hemostasis after the blood draw. Femoral veins are easier to stop bleeding, just use gauze to lightly press the blood collection site. If the blood is taken multiple times in a row, the blood collection site should be as close as possible to the centrifuge end.

　　(2) Blood collection from the external jugular vein: The syringe is pierced from the proximal end (2～3cm from the jugular vein branch) to the cephalic end in parallel to the blood vessel, so that the injection needle extends to the jugular vein branch to take blood. The blood vessels are thick here, it is easy to take blood, and the amount of blood taken is more than 10ml at a time. After the blood is taken, the needle is pulled out and the blood taken part is gently pressed with dry gauze to stop the bleeding. This method is more convenient for rabbit acute experiment.