One of the most frequently asked questions I get at lectures and from hospital transfusion services goes something like this: “What the HECK (or words to that effect) is anti-G?!” Most people have heard of the G antigen, but too few are familiar with it. So, if you’ve asked that question, don’t despair; you aren’t alone and I’ll make a G-wiz out of you yet!

A thorough understanding of G will make you a better advocate for optimal patient care, and can also be used to impress people at cocktail parties and watercoolers, as well as being a guaranteed way to put your kids to sleep at night. Trust me, you don’t want to miss this; I’m glad you’re here.

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 is different than the main group of Rh antigens (D, C, c, E, and e). G is present on any red cell that carries either the D or C antigen (or both). This means that G is only absent when a person’s red cells lack both D and C. You can see this in figure 1 below:

G presence and absence

Notice again that G is present when D is present without C, when C is present without D, and when D and C are both present in the image above.

According to the Rh genetics discussion I mentioned above (again, make sure you understand it!), a person will have G if they carry one of the following three alleles: RHD, RHCe, or RHCE. Referring to the table of Dr. Wiener’s terminology in the genetics discussion, all the haplotypes that would lead to G-positivity are in red in the table below:

D-positive HaplotypesD-negative Haplotypes
R1: DCer': dCe
R2: DcEr": dcE
R0: Dcer: dce
Rz: DCEry: 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 of those, seen in around 15% of the U.S. Caucasian population; the other two genotypes are seen in less than 1% combined). Put another way, the vast majority of G-negative people who could make anti-G are D-negative with the rr genotype.

NOTE: OK, to be completely honest, 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 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 (serine) present on the surface of both D and C antigens (encoded by the three alleles I mentioned above, RHD, RHCe, and RHCE). I have shown G “sticking out from the side” of the D or C antigen in the image above, and in figures 2 and 3 below. There is much more detail in that discovery. Out of respect for your valuable time, I will skip that esoteric discussion and 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:

G formation 1

In figure 2 above, a D-negative patient is exposed to the G antigen through interaction with a D-negative, G-positive person who carries G as a result of the action of RHCe (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 the C antigen). In this case, you would not be surprised at the formation of anti-C, but if you were unaware of G, the added anti-D specificity would make no sense!

Anti-G formation 2

In figure 3 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 an emergency or 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?

You may be thinking, “Gee, Joe, I think I get it, but why do I care? What difference does it make if a patient has a single antibody (anti-G) or TWO antibodies (anti-D AND anti-C)?” This is an excellent question.

The short answer: From the perspective of a patient who is about to be transfused, 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, almost 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 slightly longer answer: The distinction is much more important in prenatal workups and in managing a pregnant patient. Why?

First, anti-G can be associated with hemolytic disease of the fetus/newborn (HDFN), though it tends to be milder than the famous form 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, because it is too late already! (Be cautious, though, and be sure that the patient has not recently received RhIG before making that decision)
  • If the antibody is NOT anti-D and is actually anti-G, however, RhIG is indicated to protect the mother from actually forming anti-D

Knowing whether a patient has anti-D or should have RhIG to prevent future anti-D formation is an important distinction to make in determining a prenatal patient’s clinical treatment course. Seeing a patient with an apparent anti-D and anti-C and making an assumption they are not an RhIG candidate could have significant consequences if that patient has, in fact, instead, an anti-G.

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 anti-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 Walter Reed National Military Medical Center, taught me long ago that it’s a complicated process, and I remember thinking at the time that I must be just too stupid to completely understand all of the 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 using a procedure that first pulled one antibody out of the serum by using red cells with the corresponding antigen (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):

G differentiation 1

Figure 4 represents part 1 of the identification. In this example, the test serum (a.k.a., patient 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 Figure 4 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 dissociate (“elute”) 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 5 below:

G differentiation 2

The first adsorption should have selected only anti-D and anti-G, and the second deliberately uses D-negative, G-positive RBCs so that the only possible antibody out of the two remaining that could adsorb onto these cells is anti-G. Anti-G is then eluted from the cells and identified as anti-G by its activity when tested against 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, or non-reactive, with G+ cells.

Sounds simple, right?

G-differentiation is, in all honesty, not all that fun to perform in the laboratory! 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 6 and 7 showing adsorption testing of two different D-negative pregnant women:

G differentiation shortcut-patient 1

Note that in figure 6, 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).

G differentiation shortcut-patient 2

In the second example, in figure 7, 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.

Here are the take-homes:

  • G is an antigen present whenever D and/or C is present
  • Anti-G will react against D+ RBCs, C + RBCs, and D+C+ RBCs
  • Anti-G is pretty easy to manage in most transfusion patients: Almost all D-negative RBCs will be compatible
  • Identifying anti-G is most important for prenatal patients and will make all the difference in getting such a patient the RhIG they need, or saving them from an RhIG dose if not needed
  • Teasing out a patient’s antibody specificity is time-consuming, but is valuable for ensuring RhIG is used when indicated, and not unnecessarily
  • Adsorptions and double adsorptions, oh my!

I look forward to your further questions and comments on this tricky topic in the comments section below.

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