Chickens used for the supply of blood for the preparation of red blood cells should be housed separately from chickens used for other purposes. Usually they are not vaccinated with Newcastle disease vaccine. Blood from vaccinated chickens is acceptable if that is all that is available. Collect blood from more than one chicken. A collection of 1.0 mL from each of three chickens will usually give between 8 to 10 mL of a 10 percent solution of washed red blood cells. If you require a larger volume of washed red blood cells, collect more blood and adjust the volume of anticoagulant accordingly.
There are four steps in the preparation of washed red blood cells.
1. Collection of the blood.
2. Washing the red blood cells.
3. Measuring the packed cell volume.
4. Preparation of a 10 percent suspension of red blood cells for storage.
Blood is collected from the wing veins of three chickens as described in Section 7. It is mixed with an anticoagulant. Two anticoagulants are commonly used and easy to prepare:
Acid citrate dextrose (ACD) solution. Allow 1 part ACD to 3 parts blood.
Alsever's solution. Allow 1 part Alsever's to 1 part blood.
Recipes for ACD and Alsever's solution are provided in Appendix 3.
Two techniques for collection of blood into anticoagulant
1. Place 1 mL of ACD or 3 mL of Alsever's solution into a sterile bottle with a lid.
2. Bleed the first chicken and take 1 mL of blood. Immediately remove the needle from the syringe, gently push down the plunger and transfer the blood to the bottle. Replace the lid on the bottle and rotate it gently to mix.
3. Repeat Step 2 and take 1 mL of blood from a second chicken. Transfer the blood to the bottle of blood and anticoagulant and rotate it gently to mix.
4. Repeat Step 2 and take 1 mL of blood from a third chicken. Transfer the blood to the bottle of blood and anticoagulant and rotate it gently to mix.
The bottle now contains either 3 mL of blood mixed with 1 mL of ACD OR 3 mL of blood mixed with 3 mL of Alsever's solution.
1. Have the correct volume of anticoagulant in the syringe before the blood is taken. Allow 0.33 mL of ACD or 1 mL of Alsever's solution per mL of blood.
2. Bleed each of three chickens and take 1 mL of blood into the syringe.
3. After collecting the blood from all three chickens, remove the needles from the syringes and pool the blood samples in a sterile bottle with a lid.
Washing the red blood cells
After collection of the blood into an anticoagulant, the cells are washed and stored. It is best to use dextrose gelatin veronal (DGV) to wash the cells. If you do not have all the reagents to prepare DGV, use phosphate buffered saline (PBS). See Appendix 3 for the recipes.
Always treat blood gently to avoid heamolysis.
Mix gently with the anticoagulant.
Do not squirt the blood through the needle.
1. Transfer the blood to a container suitable for centrifugation.
2. Add DGV to fill the container. Mix gently.
3. Centrifuge at 500 g for 10 minutes.
4. Use a Pasteur pipette or a 10 mL glass pipette to remove the supernatant. Take care not to disturb the pellet of red blood cells.
5. Repeat Steps 2, 3, and 4 twice.
The cells have now formed a pellet after being washed three times and centrifuged. The next steps are the measurement of the packed cell volume (PCV) and preparation of a 10 percent solution of cells for storage.
Preparation of 10 percent red blood cells using a micro-haematocrit centrifuge to measure packed cell volume (PCV)
1. Suspended red blood cells in 10 mL of DGV storage solution.
2. Fill 2 micro-haematocrit capillary tubes with the blood.
3. Seal one end of each capillary tube with plasticine.
4. Place the capillary tubes in the micro-haematocrit centrifuge with the sealed ends facing the outside of the centrifuge.
5. Spin for 3 minutes.
6. Remove the capillary tubes.
7. Read the percent packed cell volume using a micro-haematocrit reader.
8. Adjust the volume of the DGV so that it equals the percentage measured. This achieves a final concentration that is equivalent to 10 percent.
Refer to the instructions provided with the micro-haematocrit centrifuge.
Example 1. If the packed cell volume measured 12 percent, add 2 mL of storage solution to the 10 mL suspension. The cells will then be suspended in a total volume of 12 mL of storage solution. This will adjust the concentration of the suspension of red blood cells to 10 percent.
Example 2. If the packed cell volume measures less than 10 percent for example 7 percent, centrifuge the suspension at 500 g for 10 minutes. Remove 3 mL from the supernatant to reduce the volume to 7 mL. Take care not to disturb the pellet. This will adjust the concentration of the suspension of red blood cells to 10 percent.
Instructions for the use of a micro-haematocrit reader
Refer to the instructions provided with the micro-haematocrit reader. This is often written on the back of the reader.
A summary of instructions:
1. Place the capillary tube in the slot so that the base line of the reader lines up with the bottom of the red blood cells in the tube.
2. Move the frame holding the tube so that the top line intersects the top of the liquid in the tube.
3. Use the handle to adjust the middle line to intersect with the top of the red blood cell pellet.
4. Read the percentage packed cell volume. This is the figure where the middle line intersects the scale.
Always look directly down at the reader when lining up the capillary tube with the lines on the reader. This will minimize errors in your readings.
Figure 14: Micro-haematocrit reader
Preparation of 10 percent red blood cells without using a micro-haematocrit centrifuge to measure packed cell volume (PCV)
There are two methods.
1. Use a micropipette or glass pipette to remove one mL of the pellet of packed red blood cells after they have been washed as described above. Add 9 mL of DGV to dilute to a 10 percent suspension.
2. Use a graduated centrifuge tube to prepare the washed red blood cells. Measure the volume of the pellet of washed red blood cells. Calculate the volume of DGV storage solution required to prepare a final suspension of 10 percent. Use a glass pipette to add or remove DGV to achieve the calculated volume.
Figure 15: Measuring packed cell volume in a graduated centrifuge tube
Use of a micro-haematocrit centrifuge and reader will give the most accurate readings. The cells are firmly packed down after centrifugation in the capillary tubes. The use of a micro-haematocrit reader reduces the error in the measurement of packed cell volume.
It is important to standardize the preparation of 10 percent red blood cells used in the haemagglutination and haemagglutination inhibition tests. This can be achieved by consistently using the same method to measure the packed cell volume each time the preparation is carried out.
Storage of 10 percent suspension of red blood cells
Store the suspension at 4°C. As the cells settle under gravity, the storage solution should remain clear. Any red colour in the storage solution indicates haemolysis of the cells and the suspension is not suitable for use and should be discarded. Normally a 10 percent suspension in DGV can be kept for one week at 4°C.
Storage of red blood cells in phosphate buffered saline.
Red blood cells can be stored as a 10 percent suspension in phosphate buffered saline. These suspensions can be expected to haemolyse after one or two days at 4°C. The red blood cells will settle under gravity. If the storage solution appears pink, the cells have started to lyse and a fresh suspension of washed red blood cells should be prepared.