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The G antigen and Anti-G

One of the most frequently asked questions I get at lectures and from testing clients goes something like this: "What the HECK (or words to that effect) is anti-G?!" If you have asked that question (even if it has been answered and you can't remember the answer!), this very long section is for you!

The Basics:

Any discussion of G has to begin with a basic understanding of Rh blood group system nomenclature and genetics. I have covered this elsewhere on the Blood Bank Guy website, so if you aren't completely clear on Rh and how the terminology and genetics work, go now and check out the details (then come back, ok?).

The G Antigen:

The G antigen, unlike the main D/C/c/E/e group of Rh antigens, is present on any red cell that carries either the D or C antigen. This means that G is only absent when a person's red cells lack both D and C, as follows:

Phenotypes with G Present Phenotypes with G Absent
D+C+
D–C+
D+C–
D–C–

According to the gene discussion referenced above, that means that a person will have G if they carry one of the following three alleles: RHD, RHCe, or RHCE. Referring back to the table of Dr. Wiener's terminology, I have placed all the haplotypes that would lead to G-positivity in red in the table below:

D Positive Haplotypes D Negative Haplotypes
R1: DCe r': dCe
R2: DcE r": dcE
R0: Dce r: dce
Rz: DCE ry: dCE

It should be pretty obvious, then, that most people are G-positive, as the only possible genotypes (combinations of the above haplotypes) that would result in someone being G-negative are rr, rr", and r"r" (rr is by far the most common combination of these three, and it occurs in around 15% of the US caucasian population; the other combinations are seen in less than 1% combined). Put another way, the vast majority of anti-G is seen in D-negative people who have the rr genotype.

NOTE: OK, to be fair, you should know that there are a very small number of D+C- people that lack G, as well as a very small number of D-C- people that have G. These are certainly the exceptions rather than the rule, but either situation can definitely happen.

Biochemically, expression of the G antigen depends on the presence of a common amino acid present on the surface of both D and C antigens. There is much more detail in that discovery, but I don't want to make your head explode, so let's move on to anti-G and its significance!

The G antibody (Anti-G):

Anti-G is an antibody formed in almost all cases by D-negative, G-negative patients with the genotype rr (dce), for reasons discussed above. The classic manner that anti-G is seen is in a D-negative patient who has never been knowingly exposed to Rh-positive blood, yet presents with an antibody that looks like a combination of both anti-D and anti-C (sometimes called "anti-CD"). This antibody can be induced either by pregnancy or transfusion, and most commonly results from exposure of an rr patient to blood from someone with either an r' or sometimes to an R0 haplotype. Two examples of this are shown below:

Figure 1

In figure 1 above, a D-negative patient is exposed to the G antigen through interaction with a D-negative-but-G-positive person who carries G as a result of the action of the RHCe allele (r' haplotype). This interaction could occur with pregnancy, and is also typical of a presentation seen after transfusion (such interaction, by the way, is completely unpreventable in transfusion, as the donor in this case would appear completely compatible with the recipient, and we don't routinely screen D-negative donors for the presence or absence of C antigen). Just glancing at the above example, you would not be surprised at the formation of anti-C, but the added anti-D specificity makes no sense unless you understand that G is present whenever either C or D is present.

Figure 2

In figure 2 above, a D-negative patient is exposed to G through interaction with D-positive-C-negative-G-positive red cells, either deliberately during a transfusion (most commonly a massive transfusion), or as a result of pregnancy. Again, a cursory examination would not leave you surprised that the patient has an antibody with anti-D specificity, but the anti-C part of the specificity is only explained by the presence of anti-G. Please note that this is only one example, and any combination of D-positive red cell exposure (whether from donors with C as well, such as those with R1 or Rz haplotypes or from those without, such as those with R0 and R2 haplotypes) could potentially lead to anti-G formation in this D-negative recipient. The difference is that you would not be surprised to find anti-C + anti-D in situations when a patient was exposed to both antigens.

Why do we care, anyway?

If you have made it this far, congratulations! You may be thinking, "Gee, Joe, the explanation makes a little bit of sense, but why do I care? What difference does it make if a patient has an anti-G or anti-D plus anti-C?" From the perspective of a patient who is about to be transfused, realistically, the distinction makes NO difference whatsoever. A patient with anti-G would be transfused in exactly the same way as a patient who has the combination of anti-D and anti-C: With D-negative blood. Remember, just about everyone who is D-negative has the genotype rr, meaning that they should be negative for D, C, and G (a transfusing laboratory would, of course, verify that the person was D- and C-negative before the transfusion).

The distinction is much more important, however, in prenatal workups. First, anti-G can be associated with hemolytic disease of the fetus/newborn (HDFN), though it tends to be milder than that caused by anti-D. Second, when a D-negative mother has antibodies that look like anti-D along with anti-C in the antibody screen that is a routine part of prenatal care, the laboratory must ask a very important question: Is this truly anti-D? The answer will determine the obstetrician's next step. If the antibody really is anti-D, the patient does not need to receive Rh Immune Globulin (RhIG) to prevent the formation of the antibody (provided the patient has not recently received RhIG). If the antibody is actually anti-G and NOT anti-D, however, RhIG is still indicated to prevent formation of anti-D. That important distinction brings us to the last portion of this discussion.

Separating anti-G from a combination of anti-C and anti-D:

If you really want to make your brain hurt, just ask an immunohematology reference laboratory technologist about how to separate anti-G from anti-C and -D; you will be running for the ibuprofen before you know it! You will hear about "double adsorptions" and "testing adsorbed eluates" and the like (my first mentor in immunohematology, the fabulous Connie Howard at the late great Walter Reed Army Medical Center, taught me long ago that I'm just too stupid to completely understand all of those details!). Let me try to make it easier for both you and I...with pictures!

Classically, immunohematologists would distinguish between anti-G and both anti-C and anti-D by the use of a procedure that pulled first one antibody out of the serum (adsorption), then isolated the anti-G by the use of a second adsorption procedure. That procedure is sometimes called "G-differentiation." Here's how it would look, using a patient that has all three antibodies (anti-D, anti-C, and anti-G):

Step 1 of G differentiation

Figure 3 represents part 1 of the identification. In this example, the test serum contains anti-C, anti-D, and anti-G. The adsorption is performed using D+G+C- RBCs (in an attempt to isolate the anti-D and anti-G and leave the anti-C behind in the adsorbed serum). In our example above, that attempt was successful, and an eluate of the red cells in this portion would contain anti-D and anti-G, but not anti-C. The next step is to elute (dissociate) the bound antibodies from the test cells and use the eluate (containing anti-D and anti-G) to perform a second adsorption (the "double adsorption" I mentioned above) with red cells of a different phenotype, as shown in figure 4 below:

Second adsorption using eluate from first adsorption

Note that D-negative, G-positive RBCs are specifically chosen so that the only possible antibody that could adsorb to these cells (out of the original three that we were evaluating) is anti-G. The anti-G is then eluted from the cells and identified as anti-G by its activity vs. C and D positive RBCs. Here is the key to understanding the two figures above: If no anti-G is present, following the same steps with only anti-C and anti-D would leave you with a final eluate that was completely negative.

G-differentiation is, in all honesty, somewhat of a pain in the posterior to do! It is time-consuming and specialized, and as a result, some reference laboratories have modified their procedures to incorporate a bit of a shortcut. Think about it: The most important question for pregnant D-negative ladies in this situation is really: "Is anti-D present?" The presence of anti-G can be assumed if anti-D is not identified. This process is sometimes called "D-differentiation," and here's how it looks, with figures 5 and 6 showing adsorption testing of two different D-negative pregnant women:

D-differentiation appearance without anti-D
D-differentiation appearance with anti-D

Note that in figure 5, no anti-D is present. The technologist would test the adsorbed serum, note the lack of anti-D, and recommend the administration of RhIG (assuming that the pattern of anti-C plus anti-D was caused by anti-G). In the second example, in figure 6, anti-D is present in the adsorbed serum and would be identified on testing that serum, so the technologist would not recommend RhIG (though the presence of anti-D opens a whole new discussion about management of the pregnancy, etc).

Gasp! Wheeze! Choke! Sputter!

Seriously, you are still here? Wow, thanks! I apologize for the length of this post, but I felt it was important to be more thorough than most regular blood bank references, because so many people don't understand this topic. I hope that this was helpful. Feel free to comment.

Thanks to Monica LaSarre and Cami Melland for helpful criticism and resource materials.

Written by DJC and originally published on the Blood Bank Guy Blog on 8/11/11; updated 11/8/12