Tag Archives: ANTISERA FOR ABO GROUPING

Requirements for Single tube compatibility technique using AHG reagent

The following are required:
1. Patient’s serum

2. Donor’s washed 3% red cell suspension
prepared as follows:

  • Transfer 0.2–0.5 ml of red cells from the pilot tube of the donor blood into about 5 ml of physiological saline  and mix. 
  • Centrifuge at high speed (e.g. 1 000 g) for about 2 minutes.
  • Discard the supernatant fluid and resuspend the cells in a further 5 ml of saline. Mix, centrifuge, and discard the supernatant fluid.                                        
  • Prepare a 3% red cell suspension by adding 1 volume of packed cells to 30 volumes of saline.

3. Antiglobulin polyspecific (Broad-spectrum) reagent, usually colour coded green. AHG reagent will agglutinate red cells sensitized with antibodies and/or coated with detectable levels of complement components.

4. Anti-D serum to make AHG control cells.

5. AHG control IgG sensitized red cells, prepared as follows:

  • Wash group O Rh positive red cells (obtained from a group O Rh positive person) three times in saline. Discard the final saline supernatant fluid.
  • Add an equal volume of IgG anti-D to the packed red cells and mix.
  • Incubate at 37 C for 30 minutes. Wash the cells four times in saline. Remove the final supernatant fluid.
  • Suspend the packed cells in saline to make a 5% red cell suspension. When added to AHG reagent, the sensitized cells should show visible agglutination
  • Store the sensitized cells at 2–8 C. They can be kept for 2–3 days.

ANT A & B SERA FOR ABO CELL & SERUM GROUPING

Whenever possible, use monoclonal blood grouping antisera. Most commercially available antisera are monoclonal. Anti-A antiserum is colour coded blue and anti-B antiserum is colour coded yellow.

Commercially produced antisera contain a preservative (e.g. 0.1% sodium azide) and most have a shelf-life of two years from the date of manufacture.

Antisera require storage at 2–8°C. Not all antisera can be frozen (this information can usually be found in the manufacturer’s literature).

When using a monoclonal anti-A antiserum it is not necessary to include an anti-AB antiserum in cell grouping.

Most monoclonal anti-A and anti-B sera are produced by tissue culture using hybridoma cell lines derived from fusing
(hybridising) mouse myeloma cells with specific antibody secreting lymphocytes (from immunized mice). The fused
cells are cloned and cultured.

Why use Monoclonal reagents?

  • They are specific, stable and reproducible, giving consistent results.
  • They agglutinate strongly rapidly (avidly) and are able to detect weak reacting antigen variants, e.g. A2 and other weak antigen A variants.
  • Being of animal origin, they are free from infectious agents such as HBV and HIV.
  • Polyagglutination due to the Thomsen phenomenon does not occur because anti-T antibody is not present in monoclonal antisera.

See also:

Blood sample for ABO grouping