Antibodies that react at 37o C are more likely to be clinically significant than those that react only at RT or lower. Unfortunately, test phases that are designated as 37o C may not always maintain 37o C, e.g., if tests are allowed to cool before reading. Also, cold antibodies may bind C3 at RT before incubation at 37o C. If this occurs, polyspecific AHG serum used for the IAT (a 37o test) will detect the C3 bound at RT. As well, reactions that happen in vitro may not always occur in vivo.
Test phases that are done at 37o C include phases that generally detect rare examples of IgM antibodies capable of reacting at body temperature (saline 37o, LISS 37o, albumin 37o), and phases that also detect IgG antibodies (enzyme 37o tests and indirect antiglobulin tests or IATs). Theoretically, antibodies reacting in any of these phases, provided the temperature has been maintained at 37o C, should be considered to be clinically significant. A notable exception relates to enzyme-only antibodies.
Antibodies that react only in enzymes at 37o C are generally considered to be clinically insignificant. This includes examples of both IgM antibodies (which are typically non-reactive at 37o) and examples of very weak IgG that typically do react at 37o . Studies have shown that such antibodies, regardless of specificity, do not cause detectable hemolytic transfusion reactions.
As mentioned earlier, cold antibodies may bind C3 at RT before tests are incubated at 37o C. If this occurs, polyspecific AHG serum used for the IAT may detect the cold antibodies. Note that this will happen only if serum from clotted patient specimens is used and only if polyspecific AHG serum is used. Of course, when an IAT is positive with polyspecific AHG serum, the result may be due to detecting warm IgG antibodies, to detecting complement bound by warm IgM or IgG antibodies, or to detecting complement bound by cold IgM antibodies. As such, the result needs to be investigated. If the cause is a cold antibody that has bound complement at RT, time and effort will have been spent on investigating a clinically insignificant antibody.
Using these criteria, antibodies in the following blood group systems are considered to be clinically significant:
Antibodies that usually are clinically insignificant include most examples of anti-M, -N, -P1, -Lea, and -Leb.
Note: The above lists do not substitute for technical and clinical judgement. Every case needs to be examined on its own merit as clinical circumstances and patient conditions differ.
Saline or LISS incubation at RT:
Incubations at RT are inappropriate because they will detect cold IgM antibodies that are clinically insignificant. Today some labs do a saline IS crossmatch at RT (instead of an electronic crossmatch) to confirm ABO compatibility between patient and donor. However, this method does not involve an incubation period.
Enzyme 37o C :
Similarly, using enzyme phases for routine pretransfusion testing is contraindicated because enzymes (despite being incubated at 37o C ) tend to detect clinically insignificant cold antibodies in addition to warm IgG antibodies. Moreover, antibodies that react only in enzymes have not been shown to be clinically significant.
Polyspecific antiglobulin serum:
Transfusion services that use polyspecific antiglobulin serum for pretransfusion tests may detect some clinically insignificant antibodies that bind C3 at RT. The trade-off is the ability to detect very weak IgG antibodies that also bind complement (see Controversies).
These methods are not used routinely, take a considerable expertise to perform, and will be discussed in principle only. Further details are available in standard reference texts.
Both the in vitro and in vivo methods below may be used to help determine clinical significance in special circumstances, including when the patient has:
The MPA is an in vitro test that can used to predict the clinical significance of allo- and autoantibodies. The method is based on the fact that splenic macrophages have receptors for IgG antibody molecules and can phagocytose red cells coated with IgG. A simplified description of the assay is as follows:
An in vivo crossmatch with
In brief, the steps in the process are as follows:
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