- Collect the blood into a glass container and allow it to clot at room temperature for 1h.
- Once the clot has formed, loosen it from the walls of the container to aid retraction.
- Keep at 4oC and leave it there overnight.
- Collect the expressed serum and centrifuge at 150 g for 5 min (to sediment erythrocytes) and then at 350g for 15 min.
- Transfer the serum (straw-colored supernatant) to containers suitable for long-term storage and heat at 56oC for 30 min to destroy the heat-labile components of complement.
- Serum may be frozen at -20oC for long-term storage but continuous freezing and thawing should be avoided.
- Protein denaturation at room temperature is minimal if serum is purified by filtration (0.22-μm pore size). For filter sterilization of volumes of serum greater than 20 ml use a combination of filters; 0.45-μm prefilter pad, 0.22-μm filter. A single 0.22-μm filter will block very rapidly.
- Alternatively, storage at 4oC is possible after the addition of merthiolate (0.01% w/v, final concentration) as a preservative agent.
- Preservation of non-sterile serum for transport without refrigeration can be achieved by the addition of 50% v/v glycerol with no interference with its immune reactivity.
For centrifugation conditions, it must be specified in terms of relative centrifugal force (RCF) which is expressed in units of gravity (times gravity or x g). Many microcentrifuges only have settings for speed (revolutions per minute, RPM), not relative centrifugal force. Thus a formula for conversion is required to ensure that the appropriate setting is used in an experiment. The relationship between the revolutions per minute( RPM) and relative centrifugal force( RCF ) is as follows:
Where g is the relative centrifugal force, R is the radius of the rotor in centimetres, and S is the speed of the centrifuge in revolutions per minute. Values of RCF in units of times gravity (x g) for common microcentrifuge rotor radii appear in the following conversion table. As an example, centrifugation of a sample at 5,000 RPM in a microcentrifuge that has a rotor with a radius of 7 cm will deliver a centrifugal force of 1,957 x g.
Differences Encountered in Real Laboratory
1.Always wear gloves and coat while performing the experiment in order to minimize exposure hazards..
2. Sterilize all the glass equipments and keep neatly in the lab table before doing the experiment.
3. You should ensure that the water bath is set to 56 degree celsius before performing the experiment.
4. All parts of the syringe coming into contact with body should be kept free of contamination.
5. Before collecting the blood, take care the individual position, ie., the person should be either sit in a chair, lie down or sit up in a bed.
6. Approach the individual in a friendly calm manner, provide for their comfort as much as possible and gain the individual cooperation.
7. Palpate and trace the path of veins with index fingers.
8. Prepare the venipuncture site using appropriate antiseptics. For example use alcohol wipes ie 70% isopropyl alcohol to clean the area. Cleanse the area in circular fashion and allowed to air dry .
9. Do not palpate venipuncture site after cleansing.
10. The needle should form a 15-30 degree angle with the surface of the arm. Swiftly insert the needle through the skin and into the lumen of the vein.
11. Remove the needle from the person's arm using a swift backward motion.
12. During blood collection to the syringe, make sure that you do 'Not replunge the syringe into the vein'. If you do so it will results in air embolism.
13. Press down on the gauze once the needle is out of the arm, applying adequate pressure to avoid formation of a hematoma.
14. Discard the used needles and syringe immediately after use which virtually eliminates transmission of blood borne pathogens.
15. Wash your hand frequently and properly after blood collection.
16. Make sure to separate serum from red blood cells within 60 minutes (1 hr) of venipuncture.
17. During the collection of serum using pasteur pipette, you should take care the tip of the pipette do not touch the lysed cells settled at the bottom of the tube.
18. Before using the syringe /Disc filters to purify the serum, ensure that you are using the correct membrane filter to optimize the flow rate and throughput.
19. Avoid repeated freezing and thawing of serum sample during its long term storage.
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