Saline tests detect IgM antibodies and can be done at 4°C, 15°C, RT, or 37°C, In general, IgM antibodies react best at 4°C. Some have a maximum range of RT (22°C), and a few can react at 37°C (body temperature) and be clinically significant.
Saline-reactive IgM antibodies can directly agglutinate red cells suspended in saline because the IgM molecule can span the gap between red cells. The test method is simple: 2 or 4 drops of serum (preferably 4 drops in order to detect weak antibodies) are incubated at the chosen temperature with 1 drop of a 3 - 5% cell suspension for 15 - 60 minutes. The test is read macroscopically for agglutination and hemolysis and, if negative, can be read microscopically. In a crossmatch, the test would be incubated at 37°C, as we only want to detect antibodies that can react in vivo at body temperature. In an antibody identification, tests could be incubated at a variety of temperatures, including 4°C, since IgM antibodies usually react best in the cold. Incubation at 4°C (or RT) would normally only be done to resolve antibodies causing an ABO discrepancy.
Antibodies detected by saline tests include: anti-M, anti-N, anti-S (sometimes), anti-Lea, anti-Leb, autoanti-I,and anti P1.
The advantages of the saline method are 1) simplicity, 2) it is the best way to detect IgM antibodies. Disadvantages include 1) it will not usually detect IgG antibodies, 2) if done at temperatures lower than 37°C, it will detect clinically insignificant IgM antibodies.
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