In general, elutions to obtain antibody for identification consist of the following steps: (1) Wash the patient's cells well six times in large volumes of saline; (2) save the last wash saline (for quality control); (3) to the washed packed cells add an equal volume of saline, or AB serum, or 6% albumin (the fluid in which the antibody will be recovered); (4) add a specified volume of the eluting chemical(s) [or place in a 56°C bath - Landsteiner method]. Some chemical methods also require a 37°C or a 56°C incubation period at this stage, and some do not. (5) centrifuge and harvest the eluate, taking care not to contaminate it with chemicals or stroma.
As stated, numerous methods exist for doing this type of elution. The main reasons for the existence of so many methods are: 1) the methods vary in their ability to elute cold and warm antibodies; 2) the methods have disadvantages, i.e., no one method is ideal; 3) the methods vary in effectiveness in the hands of different workers so that there is no universal agreement on which one is "best."
When elutions are done prior to antibody identification on the eluate, the red cells must be thoroughly washed to remove all antibody except that bound to the cells. If this is not done, you cannot be sure that the antibody in the eluate actually came off the red cells; it could be merely traces of antibody left over from the serum. Six washes with large volumes of saline are usually sufficient. Adequacy of washing is tested by examining the saline from the last wash for presence of antibody. If antibody is present, further washing must be done.
Landsteiner Heat (56°C): This method is mainly useful for cold antibodies or antibodies that react in both the cold and the warm, e.g., antibodies that cause ABO-HDN. It does not recover warm antibodies as well as the other methods but is relatively simple and rapid.
Ether: This method is well established (developed by Rubin in 1963), has good recovery for warm antibodies, and is simple and rapid to perform. It has several disadvantages related to safety (and so is not as widely used as it once was): highly flammable, requiring special handling and storage; narcotic; and toxic at high concentrations.
Acid: This method is widely used as an alternative to the ether method and is available in a commercial kit (Elu-Kit II). It gives a good recovery for warm antibodies. Disadvantages include: it is more complex than other chemical methods, requiring separate steps to add acid and phosphate buffer; the eluate may hemolyse cells in subsequent testing if the pH is not restored adequately.
Chloroform/Xylene: Both of these methods are relatively simple and rapid and have been reported to be as effective as ether and acid for the elution of warm antibodies. Xylene is highly flammable requiring special storage, and both are carcinogenic and toxic at high concentrations.
Note: None of the above methods are suitable if the purpose is to elute antibody and save the red cells for antigen typing or for autoadsorption. The heat elution at 56°C denatures antigens and the other methods hemolyse the cells.
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