Distinguishing anti-G from anti-C+D in prenatal serologic studies

A colleague asks if any institution has a policy for prenatal patients who present with what looks like anti-C+D, to prove that the antibodies are truly anti-C and anti-D, and not anti-G (as a single antibody)? Is there any value in performing this testing? Would any colleague give Rh Immune Globulin to a patient if you proved that the patient actually had anti-G and did not have anti-D?

Before sending this question to the full e-Network, a colleague at the University of Michigan was asked to comment. Here is (verbatim) what our colleague John Judd had to say "One should suspect that anti-G (and perhaps anti-C) - and not anti-C+D - is present in pregnancy whenever the titer against C+D- (r'r) RBCs is higher than the titer against C-D+ (R2R2) RBCs. This is likely due to the fact, as my departed friend John Case would opine, that the expression of G on r'r RBCs is stronger than it is on C-D+ cells. In this situation, we test the serum against the DIIIb variety of partial D RBCs, which by definition lack G. We have seen four such cases over the past few years, and all were from women who had received RhIG prophylaxis at the appropriate times. In all four cases, the DIIIb RBCs were nonreactive, and we informed the obstetricians that RhIG was indicated.

In our most recent case, the titer vs. r'r cells was 1024, but only 64 vs. R2R2 cells; the C-D+ infant did not have significant hemolytic disease. The outcome was similar to that described in the literature for most other cases. However, we should not rush to say that anti-G is an insignificant antibody with respect to HDN. There is at least one published report of anti-G in pregnancy where the infand was r'r with severe HDN.

I addressed the issue of anti-G in pregnancy in the prenatal/perinatal testing guidelines that were published in Transfusion 2001;411:445-52. Since it is unlikely that most laboratories will have access to DIIIb RBCs, another approach might be to adsorb with r'r cells, then test the adsorbed serum against C-D+ cells. Again, the resources to do such experiments will not be available to all transfusion services. Consequently, I suggest that RhIG be given if it cannot be proven that anti-D is present."

A report on the incidence of anti-G without anti-D can be found by Palfi, M et al. in Transfusion Medicine 2001;207-10."

The following responses were received.

ADDENDA Jan. 20, 2003

1. An immunohematologist at the Red Cell Immunohaematology Department, Newcastle Blood Service, in the United Kingdom suggests that colleagues might find a recently published scientific paper on the subject of anti-G versus anti-D+C to be of interest. This paper was published in December 2001 Transfusion Medicine, Volume 11, pp 443-446, and is titled "Identification and Quantification of anti-D, -C and -G in Alloimmunized Pregnant Women". The authors detail procedures followed when anti-D+C is identified, and when it is appropriate (in their opinion) to administer anti-D immunoglobulin.

ADDENDA Jan. 21, 2003

2. A Senior Technologist from Portland, Maine reports that in 1989 he presented a paper at the annual meetings of the Massachusetts Association of Blood Banks and the Maine Association of Medical Technologists about several cases of "anti-C plus anti-D" in prenatal testing. Here is a copy of the abstract that Mr. McLaughlin presented in 1989.

   DETERMINING Rh IMMUNE GLOBULIN (RhIg) CANDIDACY OF PREGNANT WOMEN WHOSE SERA APPEAR TO CONTAIN ANTI-D PLUS ANTI-C.
   "We have encountered 4 D-Du- pregnant women whose sera appeared to contain anti-D plus anti-C. In each case the initial titer of anti-C with D-C+ (r'r) RBC's was greater that the titer of anti-D with D+C- (R2R2) RBC's. In one case the antibody stimulus was traced to the transfusion of r'r RBC's. We were unable to determine the antibody stimulus in the other 3 cases. Adsorptions with r'r RBC's were performed on the 4 sera. Only 1 of the adsorbed sera contained anti-D. The other 3 adsorbed sera did not react wtih R2R2 RBC's, hence the reactions of R2R2 RBC's with the 3 corresponding neat sera were attributed solely to anti-G.
   Smith et al (Transfusion 18388, 1978) considered the advisability of administering RhIg to a woman whose serum contained anti-C plus anti-G. Issitt recommends categorizing D-Du- pregnant women as RhIg candidates if their sera contain anti-G but not anti-D (personal communication).
   In our study all 3 women whose serum contained anti-G but not anti-D recieved antenatal RhIg. One woman has delivered 2 D-Du- babies. The seCond woman recently delivered a D+C- baby and recieved RhIg. The third woman delivered a D+ baby 4 years ago and received postpartum RhIg. She is currently pregnant and does not appear to be sensitized to D.
   It is appropriate to establish RhIg candidacy of pregnant women whose sera reacts with both r'r and R2R2 RBC's by investigating whether or not anti-D is actually present. The effectiveness of administering RhIg to women sensitized to G to prevent possible sensitization to D still needs to be assessed."

Mr. McLaughlin continues saying that "we do have a procedure for adsorbing sera with apparent anti-C plus anti-D specificities from prenatal patients with Dneg, Cpos (r'r) RBC's to determine if anti-D is actually present. After 4 absorptions, the serum is tested with Dneg, Cpos RBC's to make sure the absorption was complete. If these cells react, 4 more absorptions are performed. Once the serum is completely absorbed with the r'r cells, it is tested with R2R2 RBC's. If this test is POSITIVE, anti-D is present in the unabsorbed and the absorbed sera. If this test in NEGATIVE, anti-D is absent from the unabsorbed and the adsorbed sera, and the reactivity of the D+C- red cells with the unabsorbed serum was due to anti-G. Patients whose serum contains apparent anti-C plus anti-D, but are subsequently shown not to contain anti-D by this procedure, are considered candidates to receive antenal Rh Immune Globulin, as well as postpartum if they deliver a Dpos infant." He concludes saying that "we have not had very many of these cases since 1989. I think it is important to investigate any prenatal sample with apparent anti-C+D specificity to determine if the patient is actually sensitized to D. Obtaining their transfusion history, and determining if they received D-C+ blood can be useful in solving the puzzle. The Red Cross was very helpful in retrieving donors in one case, so we could type their blood for D and C."

3. A colleague in New Zealand reports on a group O Rh negative 29 yr old female with bleeding and a positive unexpected antibody screen thought to be due to anti-C+D specificities. The patient's anti-D titre is 32 and her anti-C is 128. For exclusion of the presence of Anti-G and for the consideration of candidacy for prophylactic Rh immunoglobulin the New Zealander's lab did differential absorption and elution studies with C+D- cells and C-D+ cells with the following results:

They apparently could not come to a conclusion based on these results because of possible inadequate absorption. They decided to follow a procedure given by Mr. McLaughlin (see above). They first did the absorption of patients native sera with C+D- Group O cells until it became non reactive with C+ cells. (3 absorptions did it.) then they checked the serum with D+C- cells. They additionally ran a panel to check the specificity of the antibody present. The absorbed sera reacted strongly with D+C- cells and the identification panel clearly showed the anti-D specificity. This helped to solve their problem. One collegue challenged the interpretation commenting that if the absorbed sera does not react with D+C- cells, can one definitely say there is no anti-D, because the negative results might be due to dilution of antibody in repeated absorptions. Any comments or suggestions regarding this point is very much appreciated.

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