There are two methods for ABO grouping:
- Cell grouping (forward grouping): Red cells are tested for the presence of A and B antigens employing known specific anti-A and anti-B (and sometimes anti-A, B) sera.
- Serum grouping (reverse grouping): Serum is tested for the presence of anti-A and anti-B antibodies by employing known group A and group B reagent red cells.
Both cell and serum grouping should be done since each test acts as a check on the other.
- Autoagglutination: Presence of IgM autoantibodies reactive at room temperature in patient’s serum can lead to autoagglutination. If autocontrol is not used, blood group in such a case will be wrongly typed as AB. Therefore, for correct result, if autocontrol is also showing agglutination, cell grouping should be repeated after washing red cells with warm saline, and serum grouping should be repeated at 37°C.
- Rouleaux formation: Rouleux formation refers to red cells adhering to each other like a stack of coins and can be mistaken for agglutination. Rouleaux formation is caused by high levels of fibrinogen, immunoglobulins, or intravenous administration of a plasma expander such as dextran. Rouleaux formation (but not agglutination) can be dispersed by addition of normal saline during serum grouping.
- False-negative result due to inactivated antisera: For preservation of potency of antisera, they should be kept stored at 4°-6°C. If kept at room temperature for long, antisera are inactivated and will give false-negative result.
- Age: Infants start producing ABO antibodies by 3-6 months of age and serum grouping done before this age will yield false-negative result. Elderly individuals also have low antibody levels.
- A clean and dry glass slide is divided into two sections with a glass marking pencil. The sections are labeled as anti-A and anti-B to identify the antisera (see Figure 786.2).
- Place one drop of anti-A serum and one drop of anti-B serum in the center of the corresponding section of the slide. Antiserum must be taken first to ensure that no reagents are missed.
- Add one drop of blood sample to be tested to each drop of antiserum.
- Mix antiserum and blood by using a separate stick or a separate corner of a slide for each section over an area about 1 inch in diameter.
- By tilting the slide backwards and forwards, examine for agglutination after exactly two minutes.
Positive (+): Little clumps of red cells are seen floating in a clear liquid.
Negative (–): Red cells are floating homogeneously in a uniform suspension.
- Interpretation: Interpret the result as shown in the Table 786.1 and Figure 786.2.
Separate tubes of auto-control, positive control, and negative control should always be setup along with the test sample tube. Auto-control tube consists of mixture of patient’s red cells and patient’s own serum. This is required to rule out false-positive result due to auto antibodies in patient’s serum causing auto agglutination of patient’s own red cells. Auto-control test is particularly essential when ABO grouping is being done only by forward method and blood group is typed as AB. If there are auto antibodies in recipient’s serum, ABO grouping, Rh typing, antibody screening, and cross matching all will show positive result.
If forward grouping, reverse grouping, and autocontrol tests are all positive, then these results are probably indicative of a cold-reactive autoantibody. Before performing forward typing, red cells should be washed with normal saline to elute the antibody. Before performing reverse grouping, autoantibody should be adsorbed by washed cells till autocontrol is negative.
There are two types of cross-match: major cross-match (testing recipient’s serum against donor’s red cells) and minor cross-match (testing donor’s serum against recipient’s red cells). However, minor cross-match is considered as less important since antibodies in donor blood unit get diluted or neutralized in recipient’s plasma. Also, if antibody screening and identification is being carried out, minor cross-matching is not essential. Therefore, only the red cells from the donor unit are tested against the recipient’s serum and the name compatibility test has replaced the term cross-matching.
For transfusion of platelets or fresh frozen plasma, cross-matching is not required. However, fresh frozen plasma should be ABO-compatible.
A full cross-matching procedure consists of:
- Immediate spin cross-match at room temperature, and
- Indirect antiglobulin test at 37°C.
IMMEDIATE SPIN CROSS MATCH
- A2B donor red cells and group B recipient serum.
- Rapid complement fixation of potent ABO antibodies with bound complement interfering with agglutination.
- Rouleaux formation
- Cold-reactive antibodies: If agglutination disappears by keeping the tube at 37°C for 10 minutes, presence of cold agglutinins is confirmed.
If agglutination or hemolysis is not observed in any of the above stages, donor unit is compatible with recipient’s serum. Agglutination or hemolysis at any stage is indicative of incompatibility.
REFERENCE• Lewis SM, Bain BJ, Bates I. Dacie and Lewis Practical Hematology (9th Ed). London: Churchill Livingstone, 2001.