11.4 Recommended serological techniques for reagent testing - Reagent manufacture

11.4.1: Potency titrations

The use of a semi-automatic pipette is recommended; 1 volume being in the order of 40 µL. A separate pipette tip should be used for each reagent.

If the anti-human globulin (AHG) or other reagent is formulated with a medium to enhance its reactivity then the diluent for the determination of the potency titre should be a formulation identical to the reagent but with antibody protein replaced by non-antibody protein (e.g. fetal calf serum or bovine serum albumin.) Otherwise, dilutions may be prepared in saline containing a final concentration of 20 g/L bovine serum albumin that has not been deliberately polymerised or otherwise potentiated.

Beginning with the undiluted blood grouping reagent, doubling dilutions (e.g. 1 in 2, 1 in 4, 1 in 8) should be prepared. When preparing doubling dilutions, after the addition of the reagent or diluted reagent to an equal volume of the diluent, the tip of the pipette is emptied and blotted before the dilution is mixed and a volume transferred to prepare the subsequent dilution.

The potency titre is the reciprocal of the highest dilution of the reagent that effects a grade 2 reaction using tube and microplate or a grade 1 endpoint in column agglutination technologies.

The dilution caused by the addition of the cell suspension should not be considered in determining the potency titre.

11.4.2: Potency test methods for manual and microplate blood grouping reagents

11.4.2.1: Manual method – direct test

  1. Add 1 volume of each dilution of the reagent to a separate tube.
  2. Add 1 volume of 2–3% test red cell suspension to each tube.
  3. Mix thoroughly and incubate for the appropriate temperature and duration.
  4. Centrifuge and determine the reaction grade.

11.4.2.3: Manual method – indirect AHG test

  1. Add 2 volumes of each dilution of the reagent to a separate tube.
  2. Add 1 volume of 2–3% test red cell suspension in saline, or 2 volumes of 1.5–2% test red cell suspension in low ionic strength saline (LISS).
  3. 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.
  4. Wash the red cells 4 times.
  5. Add 2 volumes of AHG reagent to the button of test red cells and mix.
  6. Centrifuge and determine the reaction grade.

11.4.2.4: 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

  1. Add 2 volumes of each dilution of the reagent to a separate tube.
  2. Add 1 volume of 2–3% test red cell suspension in saline, or 2 volumes of 1.5–2% test red cell suspension in LISS.
  3. 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.
  4. Wash the red cells 4 times.
  5. Add 2 volumes of AHG reagent to the button of test red cells and mix.
  6. Centrifuge and determine the reaction grade.

11.4.3: Avidity determination

  1. Mix 1 volume of the undiluted reagent and 1 volume of a 30–45% red cell suspension in allogeneic serum or ABO group-compatible plasma over an oval area of approximately 20 mm × 40 mm on a glass slide.
  2. 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.
  3. Determine the time from mixing at which macroscopic agglutination first appears and record the reaction grade at 1 minute.

11.4.4: Test for IgM and IgG red cell heterospecific antibodies

These test for heterospecific antibodies which can cause haemolysis or agglutination of unsensitised red cells in the indirect antiglobulin test.

  1. Divide 12 test tubes into 2 sets of 6.
  2. Into each of the first set of tubes, add 1 volume of washed 2–3% untreated red cells in saline from 2 group A1 RhD positive, 2 group B RhD positive and 2 group O RhD positive individuals.
  3. Into each of the second set of tubes, add 1 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.
  4. Add 2 volumes of the AHG reagent, as intended to be supplied for use, to each test tube. Mix thoroughly.
  5. Incubate the reactants for 5 minutes at 19–25°C.
  6. Centrifuge the tubes and determine the reaction grade.

14.4.5: 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. The weak anti-D used for this purpose must be absorbed to remove anti-A or anti-B.

  1. To separate tubes, add 1 volume of the weak IgG anti-D to 1 volume of each of the washed 2–3% suspension of enzyme-treated RhD positive red cell samples. Mix thoroughly.
  2. Incubate for 5 minutes at 37°C.
  3. Centrifuge the tubes and determine the reaction grade.

Each of the enzyme-treated RhD positive red cell samples should be agglutinated by the weak IgG anti-D.

11.4.6: Tests for unwanted positive reactions

These test for excess anti-C3d and anti-C3c, which can cause unwanted positive reactions in the indirect antiglobulin test, and for the presence of any undesirable antibodies in the reagent.

11.4.6.1: Preparation of red cell suspensions

  1. Select integral segment lines from 2 packs of group A1, 2 packs of group B and 2 packs of group O blood stored at 2–6°C for at least 10 days.
  2. Wash each of the red cell samples with saline sufficient to remove serologically reactive traces of plasma.
  3. Prepare suspensions of each red cell sample as 2–3% in saline and as 1.5–2% in LISS.

11.4.6.2: Incubation of red cells and group-compatible serum

Each of the 6 red cell samples described above is tested as a saline and a LISS suspension with a different, fresh, group-compatible serum.

  1. For each AHG reagent to be assessed, prepare 2 sets of 6 tubes.
  2. To the first tube of the first set of 6 tubes and the first tube of the second set of 6 tubes, add 1 mL of a fresh single-donor group-compatible serum.
  3. Add 1 mL of a second fresh single-donor group-compatible serum to the second tube of each set, and so on for the 6 different fresh group-compatible sera.
  4. To the first tube of the first set of 6 tubes, add 0.5 mL of a red cell sample as a 2–3% suspension in saline. Add 1 mL of the same red cell sample as a 1.5–2% suspension in LISS to the first tube of the second set of 6 tubes.
  5. Add 0.5 mL of the second red cell sample as a 2–3% suspension in saline to the second tube of the first set of tubes and 1 mL of the same red cell sample as a 1.5–2% suspension in LISS to the second tube of the second set of tubes, and so on for each of the 6 different red cell samples.
  6. Incubate the first set of tubes (saline suspended red cell samples) for 45 minutes at 37°C. Incubate the second set of tubes (LISS suspended red cell samples) for 15 minutes at 37°C.
  7. Wash the red cell samples with saline sufficient to remove serologically reactive traces of serum. Resuspend the red cells to 2–3% in saline.

11.4.6.3: Tests with AHG reagents

  1. For each AHG reagent, prepare 2 sets of 6 tubes. To each of the first set of 6 tubes, add in sequence 1 volume of the 2–3% suspension of washed red cells from the saline test above.
  2. To each of the second set of 6 tubes, add in sequence 1 volume of the washed 2–3% suspension of washed red cells from the LISS tests above.
  3. Add 2 volumes of undiluted AHG, as supplied for use, to each of the 12 tubes. Mix thoroughly.
  4. Centrifuge the tubes and determine the reaction grade.

11.4.7: Test for anti-IgG potency

This tests the anti-IgG potency of polyspecific AHG and anti-IgG reagents used in tube or microplate techniques.

The anti-IgG reference reagent (see chapter 11.3.5) should be tested in parallel with the test reagent, each being titrated against red cells sensitised with potent IgG anti-D.

11.4.7.1: Test cells

A 2–3% suspension in saline of washed pooled group O R1r red cells is prepared from 4 individuals.

11.4.7.2: anti-D

anti-D suitable for use in this application should have a potency titre of greater than 512.

  1. To 4 mL of the potent IgG anti-D, add 2 mL of the 2–3% suspension of pooled group O R1r red cells.
  2. Mix and incubate at 37°C for 45 minutes.
  3. Wash the red cell sample with saline sufficient to remove serologically reactive traces of serum. Prepare suspensions of each red cell sample as 2–3% in saline.

11.4.7.3: Technique

  1. Prepare 1 mL volumes of 2-fold serial dilutions of the test AHG reagent and anti-IgG reference preparation from 1 in 8 to 1 in 4096 (10 tubes).
  2. Prepare a set of 10 tubes for each AHG reagent to be assessed.
  3. Place 2 volumes of each dilution into each of the series of 10 tubes.
  4. Add 1 volume of the 2–3% suspension of pooled sensitised R1r red cells to each tube and mix.
  5. Centrifuge and determine the potency titre.

11.4.7.4: Controls

The washed strongly sensitised 2–3% suspension of R1r red cells gives a negative result when centrifuged and gives negative results using the direct AHG technique with anti-complement (anti-C3c, anti-C3d, anti-C4c and anti-C4d) reagents and with AHG diluent in place of the AHG reagent.

Please note, the anti-complement specificities may be present as mixtures in 1 or more reagents.

11.4.8: anti-IgG potency by chequerboard titration studies

This tests anti-IgG potency using red cells sensitised with weak IgG antibodies (anti-D, anti-K and anti-Fya).

11.4.8.1: Selection of weak IgG antibody preparations

Antibody preparations should not be diluted to attain the following potency requirements. The use of single-donor antibody preparations is preferred.

The following are selected:

  • An IgG anti-D to give an anti-human globulin potency titre of 8–32 using a pool of group O R1r red cells from four individuals.
  • An IgG anti-K containing a final concentration of 0.014M EDTA neutralised to pH 7, to give an anti-human globulin potency titre of 8–32 using K+k+ red cells.
  • An IgG anti-Fya containing a final concentration of 0.014M EDTA neutralised to pH 7, to give an anti-human globulin potency titre of 8–32 using Fy(a+b+) red cells.

11.4.8.2: Test cells

Prepare 10 mL of a 2–3% suspension of washed R1r red cells pooled in equal proportions from 4 individuals. Similarly, prepare 10 mL of a 2–3% suspension of washed Kk red cells and 10 mL of a 2–3% suspension of washed Fy(a+b+) red cells.

11.4.8.3: Sensitisation of test cells

anti-D sensitisation

  1. Using a set of 5 containers each of 20–25 mL volume, prepare 4 mL volumes of serial 2-fold dilutions of the anti-D from undiluted to 1 in 16.
  2. Add 2 mL of the 2–3% suspension of pooled R1r red cells in saline to each container. Mix and incubate at 37°C for 45 minutes.
  3. Wash the red cells 4 times with 20 mL volumes of saline at each wash and remove the last supernatant.
  4. Add 2 mL of saline to the packed washed red cells to prepare the 2–3% suspensions of sensitised red cells.

anti-K sensitisation

As above, but using the anti-K with the K+k+ red cells.

anti-Fya sensitisation

As above, but using the anti-Fya with the Fy(a+b+) red cells.

11.4.8.4: Preparation of anti-IgG and/or AHG dilutions

For each anti-IgG and/or AHG under test, and for the anti-IgG reference preparation, prepare 2 mL volumes of 2-fold serial dilutions from undiluted (that is, as supplied for use) to 1 in 16.

11.4.8.5: Method

anti-D sensitised red cells

  1. Prepare 5 sets of 5 tubes for each AHG reagent under test and the anti-IgG reference reagent.
  2. Place 2 volumes of the AHG reagent, undiluted to 1 in 16, in the appropriate tubes for each of the 5 sets of 5 tubes.
  3. Using the 2–3% suspension of red cells sensitised with the undiluted anti-D for the first set of 5 tubes, the 2–3% suspension of red cells sensitised with the anti-D diluted 1 in 2 for the second set of 5 tubes, and so on, finishing with the 2–3% suspension of red cells sensitised using the anti-D diluted 1 in 16 for the fifth set of 5 tubes, add 1 volume of the washed red cells to each of the sets of AHG dilutions (see Table 11.4).
  4. Mix thoroughly. Centrifuge the tubes, appropriately.
  5. Determine the reaction grade.

anti-K sensitised red cells

As above, but using the anti-K sensitised K+k+ cells.

anti-Fya sensitised red cells

As above, but using the anti-Fya sensitised Fy(a+b+) cells.

Table 11.5: Chequerboard test format
Set Tube 1 Tube 2 Tube 3 Tube 4 Tube 5

1

anti-D (N)
AHG (N)

anti-D (N)
AHG (1/2)

anti-D (N)
AHG (1/4)

anti-D (N)
AHG (1/8)

anti-D (N)
AHG (1/16)

2

anti-D (1/2)
AHG (N)

anti-D (1/2)
AHG (1/2)

anti-D (1/2)
AHG (1/4)

anti-D (1/2)
AHG (1/8)

anti-D (1/2)
AHG (1/16)

3

anti-D (1/4)
AHG (N)

anti-D (1/4)
AHG (1/2)

anti-D (1/4)
AHG (1/4)

anti-D (1/4)
AHG (1/8)

anti-D (1/4)
AHG (1/16)

4

anti-D (1/8)
AHG (N)

anti-D (1/8)
AHG (1/2)

anti-D (1/8)
AHG (1/4)

anti-D (1/8)
AHG (1/8)

anti-D (1/8)
AHG (1/16)

5

anti-D (1/16)
AHG (N)

anti-D (1/16)
AHG (1/2)

anti-D (1/16)
AHG (1/4)

anti-D (1/16)
AHG (1/8)

anti-D (1/6)
AHG (1/16)

Notes on Table 11.5

N = undiluted

11.4.8.6: Controls

The unwashed 2–3% red cell suspensions sensitised with the undiluted anti-D, anti-K and anti-Fya give negative results in a spin-tube test. The washed sensitised cells should not react with the diluent or the anti-complement components of the anti-human globulin reagents.

11.4.9: Test for anti-complement potency

This tests the anti-complement potency of polyspecific AHG reagents for use in tube tests [Lachmann, 1983].

11.4.9.1: Preparation of the complement sensitised red cells

Various very low ionic strength medium techniques are used to prepare the iC3b, C4b, C3d and C4d sensitised red cells that are necessary for the assessment of anti-complement activity.

The C3 and C4 activation states produced on red cells by the various methods are shown in Table 11.5.

Table 11.5: Complement C3 and C4 activation
Method of preparation Initial state State after trypsin treatment
Very low ionic strength medium* 37°C
iC3b/C4b
iC3d/C4d
Very low ionic strength medium* 37°C
C3dg
C3d
Very low ionic strength medium* 37°C with EDTA
C4b
C4d

Notes on Table 11.5

* These media are not to be confused with LISS.

As a minimum, red cell samples from 2 individuals are to be prepared and tested as described below.

11.4.9.2: anti-C4b potency

Method

  1. Prepare a set of 3 tubes for each AHG reagent under test.
  2. Prepare doubling dilutions of the AHG reagent from undiluted to 1 in 4.
  3. Place 2 volumes of each AHG dilution in the appropriate tubes.
  4. Add 1 volume of 2–3% EC4b red cells to each tube. Mix thoroughly.
  5. Centrifuge the tubes and determine the reaction grade.

Controls

The EC4b cells do not react with anti-C3c, anti-C3d, anti-IgG or saline or the inert AHG diluent using the direct AHG technique. They react with anti-C4c and anti-C4d reagents.

11.4.9.3: anti-C4d potency

Method

  1. Place 2 volumes of undiluted AHG in a tube.
  2. Add 1 volume of 2–3% EC4d red cells. Mix thoroughly.
  3. Incubate for 5 minutes at 19–25°C.
  4. Centrifuge the tubes and determine the reaction grade.

Controls

The EC4d cells do not react with anti-C3c, anti-C3d, anti-C4c, anti-IgG or saline or the inert AHG diluent using the direct AHG technique. The undiluted AHG does not agglutinate unsensitised red cells that have been trypsin-treated, using the direct AHG technique.

11.4.9.3: anti-C3d potency

Method

  1. Prepare a set of 7 tubes for each AHG under test and the anti-C3d reference reagent (see chapter 11.3.5) which is tested in parallel, at the dilution for the 'immediate test' stated in its accompanying IFU.
  2. Place 2 volumes of each AHG dilution in each of the tubes (from undiluted, that is as intended to be supplied for use, to 1 in 64).
  3. Add 1 volume of the 2–3% EC3d/EC4d red cells to each tube. Mix thoroughly.
  4. Centrifuge the tubes and determine the reaction grade.

Controls

The EC3d/EC4d cells do not react with anti-C3c, anti-C4c, anti-IgG, saline or AHG diluent using the direct AHG technique. They do react with anti-C3d.

References

Lachmann PS, Voak D, Oldridge RG, Downie RM, Bevan PC (1983). Use of monoclonal anti-C3 antibodies to characterise the fragments of C3 that are found on erythrocytes. Vox Sanguinis, 45, 367–372. https://doi.org/10.1111/j.1423-0410.1983.tb01928.x

Last updated on 4 September 2023