Transfusion cases out of cabinet, into new book

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8. A serum sample that appears to contain anti-D and anti-C is adsorbed with R0 (Dce) RBCs. What antibodies may be present in the adsorbed serum?
a. Anti-C
b. Anti-D
c. Anti-G
d. Anti-D and anti-G
e. Anti-C and anti-G

9. Which of the following patients is eligible for RhIG?
a. A patient with anti-D
b. A patient with anti-C and anti-G
c. A patient with anti-C, anti-D, and anti-G
d. A patient with anti-C and anti-D
e. A patient with anti-D and anti-G

10. Which of the following Rh phenotypes is most likely G-negative?
a. R0R0 (Dce)
b. r’r” (CcEe)
c. r”r” (cE)
d. R1R1 (DCe)

11. Which is the correct order of potential severity of hemolytic disease of the newborn due to the following antibodies, from most severe to least severe?
a. Anti-D > anti-G > anti-C
b. Anti-G > anti-D > anti-C
c. Anti-D > anti-C > anti-G
d. Anti-C > anti-G > anti-D

12. Which of the following patients who have allo–anti-D and allo–anti-C by general antibody panel would need an adsorbtion/elution study?
a. A 65-year-old female who needs a transfusion due to symptomatic anemia
b. A 19-year-old nonpregnant female who needs blood because she was shot
c. A 26-year-old female who needs blood due to severe postpartum bleeding
d. A 75-year-old male who needs blood intraoperatively while receiving a liver transplant
e. An 18-year-old male who needs blood and was in a car accident

Case Discussion
This case describes a 28-year-old multiparous female who presented for possible RhIg after a miscarriage. The goal of this case was to describe the clinical and serologic difficulties that arise in a patient with an anti-G. Distinguishing between anti-C and anti-D versus anti-G is important in order to determine if the woman requires RhIg (to prevent anti-D formation). This scenario requires an adsorption and elution study.

Question 1 requires an understanding of forward and reverse typing. The forward type tests the recipient’s red cells with reagent anti-A, anti-B, and anti-D. The present example reveals that no agglutination was observed with any of the reagents, suggesting that the patient has no A, B, or D antigens on her red cells. This finding is confirmed by the reverse typing. Consequently, our patient has an O blood type. The reverse type does not check for anti-D in the patient’s serum as anti-D is not a naturally occurring antibody, and its presence is investigated by the red cell antibody screen (see question 2).

Question 2 tests multiple concepts. First, this question requires an appropriate interpretation of the antibody screen and antibody panel. In both the antibody screen and antibody panel, the patient’s serum (or plasma) is tested using a panel of group O red cells of known antigenic composition. The antibody screen determines the presence of a potentially clinically significant red cell antibody, and the antibody panel contains more O red cells such that the antibody specificity can be determined. In the present example, screening cell I (R1R1) and II (R2R2) are positive, indicating that a potentially significant alloantibody is present. The subsequent antibody panel reveals that the antibody is most likely an anti-D and an anti-C. This question also requires an understanding of anti-G. The G antigen is present on almost all D- or C-positive red cells and is absent from those cells that are C and D negative. The G antigen epitope is on Rh proteins (Ser103) expressing the D or C antigen. After transfusion of Rh-negative, C-positive blood, Rh-negative patients can make an anti-G, which appears to be an anti-C and anti-D on an antibody panel. Anti-G is clinically significant and is known to cause hemolytic transfusion reactions and hemolytic disease of the fetus and newborn. The antibody panel shown is not capable of differentiating anti-D and anti-C from anti-G, so additional tests are warranted in this case.

Question 3 requires an understanding of the testing required to identify an anti-G and the situation in which this testing is warranted. Sequential adsorption and elution studies are needed to determine whether anti-G is present and if anti-C and/or anti-D are also present in the patient’s serum. Since the G antigen may mask the presence of anti-D on standard antibody panels, sequential adsorption and elution studies are needed when administration of Rh immune globulin for anti-D prevention is considered important. Consequently, D-negative women who are pregnant, like the patient in this case, will need adsorption and elution studies.

Question 4 requires an understanding of the G antigen. As noted in the answer to question 2, the G antigen is present mainly on red cells with the D or C antigens. Consequently, blood that is negative for D and C antigens will also be negative for G. Moreover, as this patient may also have anti-D or anti-C in addition to anti-G, providing ABO-compatible blood that is negative for D and C antigens should provide her with the safest possible transfusion.

Question 5 requires a correct interpretation of the adsorption and elution studies, and a correct understanding of the utility of RhIg. RhIg is used in pregnant women who are at risk for becoming alloimmunized to the D antigen. Differentiating anti-G from anti-D is important in pregnancy because a woman who has made an anti-G but not an anti-D is still a candidate for RhIg prophylaxis. Panel A represents the patient’s serum sample adsorbed with r’ (Ce) RBCs. As noted above, column 1 represents the patient’s serum sample after adsorption, and column 2 represents the eluate prepared from the r’ (Ce) RBCs. Based on the reactivity shown, this patient has an anti-C and/or anti-G (agglutination seen on the eluate made by a D- C+ red cell), but no anti-D, as the serum sample (column 1) showed no agglutination when D-positive red cells were tested. As noted above, panel B represents the patient’s serum sample adsorbed with R0 (Dce) RBCs. Like panel A, column 1 represents the patient’s serum sample after adsorption, and column 2 represents the eluate prepared from the R0 (Dce) RBCs. Since panel A demonstrated that our patient had no anti-D, the reactivity from the eluate in panel B (column 2) is inferred to be from the anti-G. However, unlike panel A, the serum remaining after the adsorption shows reactivity to C-positive D-negative red cells, indicating that this patient has both anti-C and anti-G, but no anti-D. Consequently, this patient is still a candidate for RhIg.

Question 6 requires a correct interpretation of the adsorption and elution studies. See question 1 for the explanation of the correct interpretation.

Question 7 requires an understanding of the G antigen. The G antigen is present on C-positive or D-positive red cells. Consequently, cells that are negative for D and C will be negative for G. Screening specifically for G-positive or G-negative units for the crossmatch is excessively time consuming and unnecessary. Moreover, there is no commercially available anti-G typing serum available. Consequently, requesting antigen-negative units from the blood provider would be costly and impractical. Lastly, the E antigen is unrelated to the G antigen.

Question 8 requires an understanding of the G antigen and the antibodies that can form from exposure to G-antigen–positive blood. A D-negative C-negative person could form antibodies to G, C, or D. If R0 (Dce) RBCs are used for an adsorption of such a patient’s serum, then anti-D and anti-G would be removed by the R0 cell, and only a possible anti-C would remain in the adsorbed serum, which is the correct answer.

Question 9 requires a correct understanding of the utility of RhIg. RhIg is needed in D-negative pregnant women who are not yet alloimmunized to the D antigen (do not yet have anti-D). The correct answer is the only choice that does not have an anti-D.

Question 10 requires an understanding of the G antigen. The G antigen on red cells is present in individuals who are D or C antigen positive. Conversely, virtually all individuals who are D and C antigen negative are also G negative. Consequently, the person least likely to have the G antigen is someone with the r” (cE) phenotype.

Question 11 requires an understanding of the immunogenicity of the D, C, and G antigens. D is highly immunogenic, and anti-D is well known to cause severe hemolytic disease of the fetus and newborn. Anti-C is less common, and while HDFN has been reported, its effects are usually mild, and serious hemolytic disease is rare. Lastly, anti-G has been rarely reported to reach a high titer and thus is reported to be less of a threat than either anti-D or anti-C.

Question 12 requires a clinical application of the concepts addressed above. Anti-G can cause hemolysis due to an incompatible transfusion and/or HDFN. Rh-negative patients with a possible anti-G can be safely transfused with D-negative C-negative blood because the G antigen is absent from D-negative C-negative blood. Consequently, adsorption and elution studies to detect anti-G are unnecessary in most clinical situations. The only time that anti-G should be definitively evaluated is in situations where the use of RhIg is in question. As noted previously, anti-G presents as an anti-C and anti-D on standard antibody panels. Since the use of RhIg in D-negative pregnant women depends on the presence or absence of anti-D, the use of the adsorption-elution study is needed to uncover the possible underlying anti-D. Patients who are males, not of child-bearing age, or not pregnant will not need additional workup.

Reference
1. Shirey RS, Mirabella DC, Lumadue JA, Ness PM. Differentiation of anti-D, -C, and -G: clinical relevance in alloimmunized pregnancies. Transfusion. 1997;37:493-496.

Answers to questions: 1a, 2e, 3b, 4a, 5a, 6d, 7a, 8a, 9b, 10c, 11c, 12c.