Hemagglutination assay and hemagglutination inhibition test (covered in the diagnostic virology) are very important assay Virology. Both use red blood cells where some viruses tend to bind on the sialic acid receptors found their surfaces. Red blood cells need to be well prepared before being used in the two assays.


  • Whole Blood Cells
  • 50mL conical tube
  • 15 mL conical tube
  • Multichannel micropipette, micropipette, and tips
  • PBS
  • Alsever’s Solution
  • 96 round bottoms well plate
  • Microscope slide
  • Virion stock
  • Centrifuge

Collection of red blood cells Red blood cells (RBCs) used for Hemagglutination assays (HA) can be collected from chicken, turkey, piscine (fish), equine (horses), guinea pigs, and humans. The blood is collected and immediately mixed with equal volume of Alsever’s solution or in vacutainer tubes containing anticoagulant (such as EDTA) and kept at 4 °C. The blood collected in EDTA tubes must be mixed with equal volume of Alsever’s solution immediately after arrival in the laboratory.

Preparation of red blood cells (RBCs)

  1. Obtain 25mL whole blood and add 25mL cold Alsever’s Solution for anti-coagulation in a 50mL conical tube and keep on ice
  2. Centrifuge whole blood at 2,000 rpm for 10 min, 4 °C and discard the supernatant (blood plasma, buffy layer, and top erythrocytes).
  3. For washing, dilute 5 ml of RBCs in 45 ml of 1x PBS at room temperature then centrifuge at 2,000 rpm for 10 min, 4 °C and discard the supernatant.
  4. Wash 3 times by repeating step 3 above
  5. Estimate the pellet volume of RBCs by using the graduated marks on the tubes and dilute to 10% with PBS (1:10). This can be stored at 2-7˚C for up to 1 week or 42 days.
  6. Finally, the 0.5% RBC working solution for HA or HI test is prepared by diluting the RBC with PBS (1:20). The 0.5% prepared RBCs can be used for up to three days.

Why do we have to wash RBCs?

  • To obtain pure RBCs and to get rid from any other blood components such as WBCs, immune complexes, and Abs which could interfere the binding.