3.5.3 Pharmacology I
Lab 12 Collection of blood from orbital sinus of rats and separation of RBC (Cont'd...)
Step 4: Collection of whole blood: Pour 2-3% of halothane in a closed chamber and keep the animal in the chamber till anesthesia. Now, take it out of the chamber and insert the capillary tube (previously dipped into TSC solution) into the medial canthus of the eye and direct caudally and slightly dorsally. Pressure should be applied after blood collection to prevent hematomas. Collect the blood into two tubes (previously filled with TSC solution) in equal amount (1.0-1.5 ml. in each tube). Give one or two drops of antibiotic eye drops in rat's eye to avoid bacterial infection. The animal will be in the state of arousal after 15-20 minutes of the application of the anesthetics.
Fig. Orbital Sinus of a rat. Ref:http://www.theodora.com/rodent_laboratory/images/retro_orbital_sinus.jpg
Step 5: Separation of RBC from whole blood: Add NS or PBS (1:1 ratio with blood) in tubes and centrifuge at 2350 rpm for 3-4 minutes. Discard the supernatant out of tubes. Repeat the procedures for 4-5 times to wash the RBC with NS. Collect the precipitate. Because RBC is precipitated out in the tubes for it's higher molecular weight (68000).
Step 6: Dilution of RBC: Say, $X$ ml. of RBC is available after separation. Require to prepare $Y$ percent of suspension. Add $(100-Y) \times X/Y$ ml. of normal saline in $X$ ml. of RBC to get $Y$% of suspension. Collect $X$ ml. of RBC in a test tube and add the measured amount of normal saline or phosphate buffer saline into it to get the suspension.
i. The volume of collected blood should be as per the guideline otherwise the animal may die due to hypovolumic shock or physiological stress.
ii. Frequent collection should be avoided to avoid the anaemia.
iii. If an animal is required to be used for long period, then the haemacrit of the animal should be monitored to avoid the complications (haemacrit of mice, rats and rabbits are 39-49%, 36-54% 30-50% respectively).
iv. Cautiously do the anesthesia to avoid the anesthesia induced death.