ABO AND D GROUPING
ABO and D grouping must be performed by an approved technique with
appropriate controls. Before use, all new batches of grouping reagents
should be checked for reliability by the techniques used in the laboratory.
Grouping reagents should be stored according to manufacturer's instructions.
Notes:
1. Polyclonal antibodies are antibodies that are derived from different
B-cell lines. They are a mixture of immunoglobulin molecules
secreted against a specific antigen, each recognising a different
epitope.
2. Monoclonal antibodies are antibodies that are identical because they
were produced by one type of immune cell and are all clones of a
single parent cell.
ABO Grouping
Reagents for ABO Grouping
Prior to the introduction of monoclonal reagents, polyclonal anti-A, anti-B,
and anti-A,B were traditionally used for red cell grouping tests. The antiA,B reagent (from group O serum) acted as an additional check on cells that
were agglutinated by anti-A and/or anti-B but was also capable of detecting
weak A antigens (e.g., Ax). Superior monoclonal anti-A and anti-B reagents
have replaced polyclonal reagents), and although anti-A,B and anti-A+B
monoclonal reagents are available, there is no requirement to include them
in routine grouping tests. A1 and B cells are used for reverse grouping; group
O cells or an auto-control may be included to ensure that reactions with antiA and anti-B are not a result of the presence of cold autoantibodies.
D Grouping
D grouping is usually undertaken at the same time as ABO grouping for
convenience and to minimize errors that may arise through repeated
handling of patients' samples. In the absence of secure automation, testing
should be undertaken in duplicate.
Reagents for D Grouping
Monoclonal reagents do not have the problem of possible contamination
with antibodies of unwanted specificities, as was the case with polyclonal
reagents.
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Methods
There are several techniques available for ABO and D grouping including
tube, slide, microplates, and columns. Other techniques for blood grouping
have been described (e.g., flow cytometry),[21] but they are not in routine use.
Care should be taken to use the appropriate reagent because not all reagents
have been validated by the manufacturer for all techniques.
1. Tube and Slide Tests
1. Spin-tube tests are commonly used, particularly for urgent testing,
where small numbers of tests are performed at once.
2. Slide or tile techniques are widely used in under-resourced countries
for ABO and D grouping.
3. Spin-tube tests should be performed in 75 × 10 or 75 × 12 mm plastic
tubes. Immediate spin tests may be used in an emergency, whereas
routine tests are usually left for 15 min at room temperature (about
20°C) before centrifugation for 1 min at 150 g.
4. Equal volumes (1 or 2 drops from either a commercial reagent dropper
or a Pasteur pipette) of liquid reagents or serum/plasma and 2% cell
suspensions are used.
5. The patient's red cells (diluted in phosphate buffered saline [PBS])
should be tested against monoclonal anti-A and anti-B grouping
reagents.
6. The patient's serum or plasma should be tested against A1 and B
reagent red cells (reverse grouping).
7. In addition, the serum or plasma should be tested against either the
patient's own cells or group O cells (i.e., a negative control) to exclude
reactions with A and B cells as a result of cold agglutinins, other than
anti-A or anti-B, in the patient's sample.
8. Mix the suspensions by tapping the tubes, and leave them undisturbed
for 15 min.
9. Agglutination should be read as described on.
10.Any discrepancy between the results of the red cell grouping and the
reverse grouping should be investigated further, and any repeat tests
should involve cells taken from the original sample rather than the
prepared suspension.
11.Reverse grouping is not carried out for infants younger than 4 months
of age because the corresponding antibodies are normally absent.
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2. Slide Method.
In an emergency, rapid ABO grouping may be carried out on slides or tiles.
The method is satisfactory if potent grouping reagents are used. An
immediate spin-tube test is preferable.
3. Liquid-Phase Microplate Methods.
4. Column Agglutination Techniques.
5. Solid-Phase Techniques.
Controls
Positive and negative controls should be included with every test or batch of
tests. The anti-A reagent should be tested against group A (positive control)
and B (negative control) cells, and the anti-B reagent should be tested
against group B (positive control) and group A (negative control) cells.
1. All the reagents should reach the room temperature before performing
of the test.
2. The control samples should be treated in the same way as the test
samples.
3. The required controls are shown in Table 20.2 . Where controls do not
give the expected reactions, investigations should be undertaken to
determine the validity of all tests undertaken subsequent to the most
recent valid control results.
Table 20.2 -- Control cells for blood grouping
Reagent Positive control Negative control
Anti-A A cells
B cells
Anti-B B cells
A cells
Anti-D D positive cells D negative cells
A1 cells Anti-A
Anti-B
B cells Anti-B
Anti-A
Causes of Discrepancies in ABO/D Grouping
False-Positive Reactions
1- Rouleaux: The stacking of red cells on top of one another in columns is
known as Rouleaux formation.
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Rouleaux may occur in various clinical conditions, where the ratio of normal
albumin to globulin is altered in plasma (e.g., multiple myeloma), and in the
presence of plasma substitutes such as dextrans. Rouleaux will usually
disperse on a slide if a drop of saline is added; alternatively, the reverse
grouping can be repeated using plasma/serum diluted 1 in 2 or 1 in 4 with
saline.
2- Cold Autoagglutination and Cold Reacting Alloantibodies
3. Contaminating Antibodies
4. In vitro Bacterial Contamination
False-Negative Reactions
1. Failure to Add Reagents
The use of colour-coded reagents for ABO grouping is helpful in this respect.
2. Loss of Potency
Inappropriate storage or freezing and thawing may cause a loss of potency of
blood grouping reagents.
3. Failure to Identify Lysis
In the presence of complement, anti-A and anti-B may cause in vitro lysis of
reagent red cells. If lysis is not recognised as a positive reaction, falsely
negative results may be recorded in reverse grouping tests. To avoid this,
reverse grouping cells should be resuspended in EDTA saline, where serum
rather than plasma is used.
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