Bleeding of Animals and Serum Preparation

1. Bleeding of Animals and Serum Preparation Lab 3

2. objectives After completing this laboratory exercise students should be able to: • Bleed experimental animals via various routes. • Prepare serum from whole blood.

3. Blood volumes in domestic animals

4. Worth to know • Bleeding of experimental animals is needed to prepare normal serum and immunized serum as well as to obtain blood cells. • Successful bleeding requires: • Care. • Patience. • Practice. • To reduce the quantity of lipid in serum, the animals should be fasted for 12 hours prior to bleeding. • Animals can be bled by: • Cardiac puncture. • Venous Puncture.

5. Whole blood is composed of a cellular component and plasma. Upon clotting of fresh blood, the soluble fibrinogen in the plasma is converted into insoluble fibrin and the remaining fluid is the serum which is clear yellowish fluid. Although fibrinogen forms only 3% of the plasma it interferes with many types of serological tests. Serum is prepared in four steps: Coagulation, separation, clarification and preservation. It is essential to use very clean glassware and plastic ware to prepare and store sera. Sterile equipments are preferable. Turbidity of serum is a sign of high lipid content which must be clarified as lipids interfere with the visibility of antigen –antibody reaction in vitro and are sources of emboli, when serum is injected into animals. Serum vs plasma

6. Cardiac puncture - Rabbits • Secure the rabbit in supine position on a rabbit board. To ensure immobilization, you may warp a piece of gauze under the board and over the abdomen of the rabbit and tie it securely. • Anaesthetize the rabbit with Ketamine hydrochloride which act as a short- term surgical anesthesia. Inject 0.3 ml/kg of body weight intramuscularly using a 24 gauge needle (this step is optional) • Clip the hair over the thorax and about 5 cm below the sternum, wipe the shaved area with 70% alcohol. • Have a 20 or 50 ml syringe fitted with 18-gauge needle ready for heart puncture. • Touch the rabbit around the xiphoid process and the last animal sternal rib to the left side of the midline with your finger, and feel the heart beet. Hold the syringe at 30 angle and insert the needle at the junction to the left of the xiphoid process and last sternal rib while pulling the plunger. Slowly advance the needle until you feel the pulsation of the heart. Continue advancing the needle slowly and watch for blood flow into the syringe. Stop advancing the needle and hold the needle in position until the desired volume of blood has been drawn. (up to20-50 ml can be drawn from large rabbit without adverse reaction) • Withdraw the needle slowly, remove the needle, and empty the blood into a centrifuge tube.

7. Cardiac puncture - Rats • Is often used to obtain a large volume of blood to prepare serum from immunized animal after the immunization is complete. • Anesthetize the animal in etherization jar. Place a piece of cotton or gauze soaked with ether in the jar. Place the animal in the jar and cover the lid. Wait few minutes until the animal has lost consciousness. • Place the rat on a small –animal board in supine position. Fasten each leg with a piece of string. • Use a 22 gauge needle fitted to a 5 ml syringe and follow the steps of cardiac puncture of rabbits. (about 5 ml of blood can be drawn from 200 g rat without adverse effect.

8. Cardiac puncture - Mice • Anesthetize the animal with ether or Nembutal. The anesthetizing dose of Nembutal is about 0.5 mg per 10g body weight injected SC or IP. • Place the mouse on cork board in supine position and tie each foot. • Using 1ml or 2ml syringe with 26-gauge needle, follow the steps outlined for cardiac puncture of the rabbit. (around 0.5ml blood can be collected from 25 g mouse).

9. Venous puncture - Rabbits • Restrain the animal in a rabbit box. • Expose the inner or outer marginal vein of one ear by shaving the skin. • Place the inner surface of the ear above a heated 15-watt lamp bulb to help dilate the vein. Alternatively, you can dilate the vein by rubbing the ear with a gauze soaked with xylene. • Cut the vein either transversely or longitudinally using sharp sterile razor. • Hold the top of the ear in horizontal position and allow the blood to drip into a centrifuge tube. If the blood stops flowing, flick the vein with your finger. UP to 40 ml of blood can be collected from one relaxed rabbit. • Stop blood flow from the vein by placing a piece of cotton over the cut and pressing the area between your thumb and index finger. Apply a paper clip to keep the piece of cotton in place.

10. Venous puncture - Mice • Warm the mouse by placing the animal in a jar and exposing the jar to 100-watt bulb for 10-15 minutes. • Anesthetize the mouse with ether. • Cut off about 0.5 cm of the mouse tail. • Hold the mouse in a vertical position by the skin of its back and allow the blood that drips from the tail cut to collect against the wall of a centrifuge tube. Smoothly rub the tail of the mouse to enhance blood flow. Collect up to 0.5 ml blood. • Apply a piece of gauze to the cut surface of the tail and press until bleeding stops.

11. Preparation and Preservation of serum • Serum preparation requires freshly drawn blood without the use of anticoagulants. • Allow freshly drawn blood to form a clot by leaving at room temperature for 15-60 minutes. • Dislodge the clot from the wall of the tube using disposable wood applicators. • Centrifuge the blood at 3000 rpm for 5 minute. • Remove serum with pipette to eppindorf tubes carefully (0.1 - 1 ml) and store at -20°C or below. To preserve complement activity and IgE antibodies store the serum at -70°C or below. Lyophilized serum powder can be store at 4°C.

12. Preparation and Preservation of serum • To store the serum for long periods add a preservative to kill microorganisms or to arrest their growth by adding one of the following serum preservatives at the following final concentrations • Merthiolate 0.001% • Sodium azide 0.1% • Phenol 0.25% • Blood collected in non-heparinised micro capillary tubes can be used for preparing small quantities of serum using hematocrit centrifuge.