The dilution caused by the addition of the cell suspension should not be considered in determining the potency titre.
b) Potency test methods for manual and microplate blood grouping reagents
I - Manual method. Direct test
Add one volume of each dilution of the reagent to a separate tube.
Add one volume of 2–3% test red cell suspension to each tube.
Mix thoroughly and incubate for the appropriate temperature and duration.
Centrifuge and determine the reaction grade.
II - Manual method. Indirect anti-human globulin test
Add two volumes of each dilution of the reagent to a separate tube.
Add one volume of 2–3% test red cell suspension in saline, or two volumes of 1.5–2% test red cell suspension in LISS.
Mix thoroughly and incubate at 37°C for 45 minutes if the red cells are suspended in saline, or for 15 minutes if suspended in LISS.
Wash the red cells four times.
Add two volumes of anti-human globulin reagent to the button of test red cells. Mix. Centrifuge and determine the reaction grade.
III - Microplate method
Equipment
Rigid polystyrene microplates with 'U' shaped wells.
Centrifuge with microplate carriers having a radius of at least 10 cm.
Microplate shaker.
Concave microplate reading mirror or automated plate reader.
Red cells for microplate use, bromelin-treated if required.
Method
Using a microplate, add one volume (25–50 µl) of each dilution of the reagent to one volume of 2–3% test red cells.
Mix the contents of the wells using a microplate shaker. Incubate at 19–25°C for 15 minutes.
Centrifuge the microplate at l00g for 40 seconds. Gently dislodge the red cells from the bottom of the wells using a microplate shaker.
Determine the reaction grade using a concave mirror or automatic plate reader.
c) Avidity determination
Mix over an oval area of approximately 20mm x 40mm on a glass slide, one volume of the undiluted reagent and one volume of a 30–45% red cell suspension in allogeneic serum or ABO group-compatible plasma.
Maintain the slide at the recommended temperature for a slide test. If a range of incubation temperatures is given, for those blood grouping reagents where the antibody-antigen reaction is favoured by a colder temperature, the higher temperature should be used; for other blood grouping reagents, the lower temperature should be used.
Determine the time from mixing at which macroscopic agglutination first appears and record the reaction grade at one minute.
d) Test used in performance evaluation and batch release testing of AHG
i) Tests for IgM and IgG red cell heterospecific antibodies
These test for heterospecific antibodies which can cause haemolysis or agglutination of unsensitized red cells in the indirect anti-globulin test.
Method
Divide 12 test tubes into 2 sets of 6.
Into each of the first set of tubes, add one volume of washed 2–3% untreated red cells in saline from two group A1 RhD positive, two group B RhD positive and two group O RhD positive individuals.
Into each of the second set of tubes add one volume of washed 2–3% enzyme-treated red cells (papain, bromelin or ficin) in saline from the same group A1 RhD positive, group B RhD positive and group O RhD positive individuals.
Add two volumes of the anti-human globulin reagent as intended to be supplied for use, to each test tube. Mix thoroughly. Incubate the reactants for five minutes at 19–25°C.
Centrifuge the tubes.
Determine the reaction grade.
ii) Control of enzyme treatment
Weak IgG anti-D, known to be reactive with enzyme-treated red cells should effect a positive reaction with each washed, enzyme-treated, red cell sample by the following method:
To separate tubes, add one volume of the weak IgG anti-D to one volume of each of the washed, 2–3% suspension of enzyme-treated, RhD positive red cell samples. Mix thoroughly. Incubate for five minutes at 37°C. Centrifuge the tubes. Determine the reaction grade.
The weak anti-D used for this purpose must be absorbed to remove anti-A or
anti-B.
Each of the enzyme-treated RhD positive red cell samples should be agglutinated by the weak IgG anti-D.
iii) Tests for unwanted positive reactions
These test for excess anti-C3d and anti-C3c, which can cause unwanted positive reactions in the indirect anti-globulin test, and for the presence of any undesirable antibodies in the reagent.
Method for preparation of the red cell suspensions from segmented bleed line samples
Select integral segment lines from two packs of group A1, two packs of group B and two packs of group O blood stored at 2–6°C for at least ten days.
Wash each of the red cell samples with saline sufficient to remove serologically reactive traces of plasma.
Prepare suspensions of each red cell sample as 2–3% in saline and as 1.5–2% in LISS.