Clinical Significance

Given that we want to detect only those unexpected antibodies that are clinically significant, i.e., can cause in vivo destruction of transfused red cells, what mechanisms does the transfusion service have to determine clinical significance? Several mechanisms are briefly discussed below. Unfortunately, they are rather inexact.

Reactivity temperature and phase

Antibodies that react at 37^o C are more likely to be clinically significant than those that react only at RT or lower. Unfortunately, test phases that are designated as 37^o C may not always maintain 37^o C, e.g., if tests are allowed to cool before reading. Also, cold antibodies may bind C3 at RT before incubation at 37^o C. If this occurs, polyspecific AHG serum used for the IAT (a 37^o 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 37^o C include phases that generally detect rare examples of IgM antibodies capable of reacting at body temperature (saline 37^o, LISS 37^o, albumin 37^o), and phases that also detect IgG antibodies (enzyme 37^o tests and indirect antiglobulin tests or IATs). Theoretically, antibodies reacting in any of these phases, provided the temperature has been maintained at 37^o C, should be considered to be clinically significant. A notable exception relates to enzyme-only antibodies.

Antibodies that react only in enzymes at 37^o C are generally considered to be clinically insignificant. This includes examples of both IgM antibodies (which are typically non-reactive at 37^o) and examples of very weak IgG that typically do react at 37^o . 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 37^o 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.

History of causing in vivo red cell destruction

Antibodies are clinically significant if they can destroy red cells in vivo. Evidence of in vivo red cell destruction occurs in hemolytic disease of the newborn, hemolytic transfusion reactions, and experimental studies demonstrating shortened survival of transfused red cells (see below).

Using these criteria, antibodies in the following blood group systems are considered to be clinically significant:

ABO (anti-A and anti-B)
Rh (anti-D, -C, -E, -c, -e, and most others)
Kell (anti-K, -k, and most others)
Kidd (anti-Jk^a and anti-Jk^b)
Duffy (anti-Fy^a and anti-Fy^b)
MNSs (anti-S, -s)

Antibodies that usually are clinically insignificant include most examples of anti-M, -N, -P₁, -Le^a, and -Le^b.

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.

Practical implications

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 37^o C :

Similarly, using enzyme phases for routine pretransfusion testing is contraindicated because enzymes (despite being incubated at 37^o 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).

Red cell survival studies

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:

an unidentified antibody that reacts with all donor red cells tested
a known antibody that reacts with an antigen of high frequency and the antibody's clinical significance is debatable

Mononuclear phagocyte assay(MPA)

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:

Mononuclear cells from volunteers (or the patient) are collected and adhered to a coverslip or other device, e.g., tissue culture slide.
Donor or screen cells are sensitized with the patient's antibody and then incubated with the monocytes adhered to the coverslip.
After rinsing, the preparation is fixed, stained (e.g., Wright or Giemsa), and observed for rosetted or phagocytosed red cells.
The number of phagocytosed red cells per 100 monocytes/macrophages is counted and compared to counts for positive and negative controls.

⁵¹ Cr in vivo "crossmatch"

An in vivo crossmatch with ⁵¹ Cr-labelled red cells may also be helpful for determining clinical significance in the special circumstances listed above.

In brief, the steps in the process are as follows:

An aliquot of donor red cells are tagged with ⁵¹ Cr and a small volume (1 mL) is injected into the patient.
Patient blood samples are collected at intervals (e.g., at 3, 10, and 60 minutes).
The count done on the 3-minute blood sample is used as the baseline (100% survival) to calculate red cell survival at 10 and 60 minutes. Radioactivity counts are also done on separated plasma.
If the 60-minute survival is at least 70% and the plasma contains no more than 5% of the radioactivity injected, it is assumed that the entire unit of red cells will not be rapidly destroyed and cause a severe transfusion reaction.

Issuing Blood Products

Clinical Significance

Prewarm Technique