case study [transfusion medicine | immunology | hematology] Drug History

Enoxaparin, switched to heparin at 36 weeks of gestation;

Is It Really Anti-D and Anti-C nitrofurantoin; iron supplementation; and prenatal vitamins.
or Is It Anti-G?
Nada Rikabi, MD,1 Lisa Dunn-Albanasse, MD,2 Obstetrical History
Dave Krugh, MT(ASCP)SBB,1 Diane Snider, MT(ASCP),1 [T2]
Kristi Frenken, RN,2 Karen Rossi, RN,2
Richard O’Shaughnessy, MD,2 Melanie S. Kennedy, MD1 Principal Laboratory Findings
Departments of 1Pathology and 2Obstetrics & Gynecology, Maternal- [T3]
Fetal Medicine, The Ohio State University, Columbus, OH
Results of Additional Diagnostic Procedures
DOI: 10.1309/VQVYA7WY3T8HH7P4
An amniocentesis was performed at 20 weeks gestational age
(G.A.) and a delta OD450 analysis performed on amniotic
Patient fluid yielded a value of 0.07 (with a hemoglobin peak present
36-year-old Caucasian woman. in the scan), corresponding to a Liley chart zone of 1 (ie, un-
affected or mildly affected fetus for intrauterine hemolysis).

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History of Present Illness
The patient is a G3P3, group B Rh(D) negative woman,
treated appropriately with Rh(D) immune globulin (RhIg)
therapy after the birth of her first and second infants. The pa-
tient presented in her third pregnancy at 12 2/7 weeks of ges-
tation with a positive antibody screen for anti-D and anti-C.
The titer against R1R1 cells initially was 1:8, increasing to
1:16. The anti-D titer against R2R2 cells was 1:4.
Medical History
Thrombotic thrombocytopenia purpura (TTP), treated on
multiple occasions with plasma exchange and with plasma
infusions [T1], deep venous thrombosis, supraventricular
tachycardia, and adenomatous polyps of the small bowel.
Surgical History
Splenectomy.
Family History
Divorced, remarried with 3 children. The patient is adopted and
therefore had no knowledge of her family’s medical history.
Fetal cells in the amniotic fluid were tested by polymerase
chain reaction (PCR) for gene sequences corresponding to Rh
antigens. PCR testing was positive for D and negative for C
antigen gene sequences. At 24 weeks and 2 days G.A. (ie, 24
2/7), anti-G and anti-C were identified in the patient’s serum.
At 27 6/7 weeks G.A., RhIg was administered to the mother.
Questions:
1. Should all prenatal patients presenting with possible anti-D
and anti-C in their serum be routinely evaluated to deter-
mine the presence of anti-G rather than anti-D?
2. How can anti-G be identified in the laboratory?
3. What is the risk for hemolytic disease of the newborn (HDN)
in prenatal patients with anti-G as opposed to anti-D?
4. In this case, if the infant required an intrauterine transfu-
sion or an exchange transfusion, what type of blood would
be appropriate for transfusion?
5. How could the identification of anti-G in a prenatal patient
with an apparent anti-D and anti-C be of help in some
legal issues?
6. Is the patient a Rh(D) immune globulin (RhIg) candidate?

Patient’s Transfusion History

Year/Month of Transfusion and Number of Units Transfused

T1
Blood Component 1992 1993 1994 1996 1997 1998 1999 2000 193
Transfused Aug Oct Dec Jan Feb Apr May Feb Feb Jul Aug Sep

Platelet pheresis units 1a 0 0 0 0 0 0 0 0 0 0 0

Pooled platelets 1a 0 0 0 0 0 0 0 0 0 0 0
FFP 12 43 0 0 14 2 2 0 0 0 0 0
CPP 0 0 6 6 124 62 12 91 2 8 80 0
RBCs 4a + 1b 0 0 0 2a 2a 0 1a 2a 0 2b 3a
aB Rh(D) negative. bO Rh(D) negative. FFP, fresh frozen plasma; CPP, cryoprecipitate-poor plasma; RBCs, red blood cells

© laboratorymedicine> march 2003> number 3> volume 34

 Patient’s Obstetrical History

Mother Neonate(s)a
T2
Pregnancya Ab Screen G.A. @ Delivery RhIg Givenb Probable HDN Status Rh(D) Type
Paternal Genotype

G1P1 Neg 40 Yes n.a. Unaffected Positive

G2P3 Neg 40 Yes n.a. Unaffected Negative (twins)
G3P4 Pos 39 Yes R2r Unaffected Positive
aEach pregnancy resulted from 3 different men. bRh(D) immune globulin (RhIg) was administered at ~28 weeks gestational age (G.A.) or at delivery for all three pregnancies. Ab,

antibody; HDN, hemolytic disease of the newborn; n.a., not available

T3
Patient’s Antibody Screening Results at Various Gestational Ages

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Antibody Titer at Gestational Age, wk

Ab Testing Phasea 12 2/7 16 3/7 22 22 25 2/7 27 6/7 32 1/7

Saline Neg Neg Neg Neg Neg Neg Neg

AHG 1:8 1:16 1:4 1:16 1:16 1:8 1:8
aAntibody (Ab) screening cells were genotype R1R1 for all maternal serum Ab screening performed at all gestational ages (G.A.) except at 22 weeks G.A. when both R1R1 and R2R2 cells
were used. AHG, anti-human globulin

Possible Answers: ing by adsorption/elution techniques in the presence of poly-

1. Yes. Routine evaluation to differentiate anti-D, anti-C, and
anti-G should be done on alloimmunized pregnant women
presenting serologically with an apparent anti-D and anti-C in
their serum. Anti-G should be suspected when (1) the obstet-
rical and transfusion history conflicts with formation of anti-
D and -C, (2) the pregnant woman has received appropriate
Rh(D) immune globulin (RhIg) therapy, (3) the biological
father of the infant is Rh(D) negative, and/or (4) the antibody
titer of anti-C is significantly higher than the anti-D titer. In
one case report, the laboratory investigation of a G7P3
woman, who received appropriate RhIg therapy after her last
pregnancy, presented with a positive antibody screen with an
apparent anti-D and anti-C. Anti-G was suspected because
more reactivity was observed with the C(+)D(-) red blood
cell sample than with the C(-)D(+) red blood cell sample.
Further testing with D(+)G(-) red blood cells showed no reac-
tivity, leading to the exclusion of anti-D.1 In another study, a
G4P1 pregnant woman who had received RhIg therapy when
indicated was shown to have anti-D and anti-C with a higher
194 titer of anti-C than anti-D (1:32 versus 1:4, respectively). Ad-
sorption and elution studies proved that the patient had anti-C
and anti–G, but no anti-D. Retrospective analysis of sera
from 6 other pregnant women with anti-D and anti-C was
done; 2 had anti-D, anti-C, and anti-G, 3 had anti-D and anti-
G with no anti-C, and 1 had anti-C and anti-G with no anti-
D.2 In another report, 27 pregnant women who were initially
identified as having anti-D and anti-C underwent further test-
ethylene glycol (PEG) using R0r, r’r, and rGr red blood cells.
Anti-C and anti-G with no anti-D were noted in 4 of 27
(14.8%) samples.3 In our case, anti-G was suspected due to
higher titers of anti-D and anti-C compared with anti-D, as
well as the lack of history of sensitization [all red blood cells
transfused were Rh(D) negative and RhIg was administered
appropriately when indicated]. Since the differentiation of
anti-D and anti-G is important in the management and prog-
nosis of a pregnancy complicated by the presence of these
antibodies, it should be performed whenever anti-D and anti-
C are identified.
2. By testing serum with an appropriate panel of genotypic
red blood cells. The G antigen is present on all red blood
cells that are C+, since C(+)G(-) red blood cells have not
been found. The G antigen is present also on most (not all) D
antigen positive red blood cells, since D(+)G(-) red blood
cells have been found. The G antigen (weak) is present on
Rh(D) positive weak red blood cells. In summary, the G anti-
gen would be absent from C(-)D(-) red blood cells and would
be present on most C+ or D+ red blood cells. Because of the
way these 3 antigens are expressed, anti-G appears serologi-
cally identical to anti-D and anti-C. In a pregnant patient with
an apparent anti-D and anti-C, sequential adsorption and elu-
tion studies using r’r (Cde) and R0r (cDe) red blood cells can
be done to confirm the presence or absence of anti-G [T4]. It
is important to mention that rarely, individuals may have red

laboratorymedicine> march 2003> number 3> volume 34 ©

Antibodies Remaining After Sequential Reaction of Serum Containing Anti-D, Anti-C, and/or Anti–G
Antibodies With R0r and r’r Genotype-Specific Red Blood Cells
Antibodies Remaining After Reaction With R0r and r’r Cells
R0r (cDe, G positive)
Serum Containing the Antibodies 1st Adsorption ➙
D, C, and G D and G
D and C D D
blood cells that are D negative, C negative, but G positive.
These cells may have a C-like component known as (CG) that
can react with anti-C. Because of the possibility of this reac-
tion, these rare cells should not be used to detect the presence
or absence of anti-G.2
3. The risk of HDN is small but finite. The number of case
reports of anti-C and/or anti-G without a concomitant anti-
D during pregnancy is limited, precluding a complete evalu-
ation of the risk for HDN in these patients. However, it is
known that anti-C alloimmunization occurs less frequently
than anti-D and even though anti-C may cause serious
HDN, it is usually less severe. Anti-G rarely presents with a
high titer that may affect the fetus and require medical in-
tervention (ie, amniocentesis or intrauterine transfusion). In
one study, adsorption and elution testing was performed on
serum from 27 pregnant women with apparent anti-D and
anti–C and 4 of the 27 samples (14.8%) showed anti-G and
anti-C without anti-D. None of the newborn children from
these pregnancies were affected with HDN or needed post-
partum management.3 In another case report, a 35-year-old
Rh(D) negative primigravida who had received 4 units of
Rh(D) negative allogeneic red blood cells had a history of
positive anti-D and anti-C antibody screens as part of her
prenatal workup. Additional studies by a different labora-
tory showed the presence of anti-G and possible anti-C with
no anti-D. At 22 weeks gestation, titers were 1:64 against r’r
red blood cells and 1:16 against R2R2 red blood cells. Am-
niocentesis indicated that the fetus did not appear to be af-
fected by intrauterine hemolysis. The mother delivered a
healthy infant at 36 weeks gestation. The infant had a posi-
tive direct antiglobulin test (DAT) with an acid eluate
revealing the presence of anti-G. The total bilirubin (highest
value) was 11.9 mg/dL at 5 days postpartum and no further
management was needed.4 In contrast to these findings,
among 28 sera from alloimmunized women with anti-D and
anti-C, 2 contained titers of anti-G consistent with moderate
to severe HDN by the chemiluminescence test. It was also
noted in this study that fetal hemolysis due to anti-G was
more likely to occur in r’r (C-positive, D-negative) fetuses.5
In another study, an Rh(D) negative woman with a history
of multiple transfusions of Rh(D) negative blood in the past
© laboratorymedicine> march 2003> number 3> volume 34
T4
r’r (Cde, G positive)
1st Elution ➙ 2nd Adsorption ➙ 2nd Elution
D and G G G
None None
was thought to have anti-D and anti-C. Prenatal workup re-
vealed that the patient had an anti-G rather than an anti-D.
At 32.5 weeks of gestation, the mother delivered an affected
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fetus with moderately severe HDN.6 We conclude that even
though hemolytic HDN due to anti-G is very uncommon, it
should still be considered when more reactivity is observed
with C(+)D(-) than with the C(-)D(+) antibody screening
red blood cells.
4. Cytomegalovirus (CMV) seronegative, irradiated group O
Rh(D) negative (rr) red blood cells would be indicated. The
transfused red blood cells must be D and C antigen negative
and consequently G antigen negative. The differentiation of
anti-D, anti-C, and anti-G for routine transfusion is not nec-
essary, since most Rh(D) negative, C antigen negative red
blood cells would be G antigen negative. If the mother is the
donor for intrauterine transfusion, frozen deglycerolized red
blood cells allows multiple aliquots to be frozen and then
thawed as needed.
5. When the medical and transfusion history are inconsistent
with the finding of an apparent anti-D and anti-C in an Rh(D)
negative pregnant woman and the biological father is Rh(D)
negative. In such cases, questions about paternity may arise.
If anti-D is not present (only anti-C and/or anti-G) RhIg
should be given at appropriate times. Therefore, correct anti-
body identification and appropriate RhIg administration are
extremely important in preventing medicolegal situations.3
6. Yes. Pregnant women with anti-G and without anti-D in
their serum are considered candidates for prophylactic RhIg
therapy at 28 weeks gestation, at delivery if the infant is
Rh(D) positive, and when clinically indicated (eg, for the risk
of sensitization with amniocentesis) to prevent formation of
anti-D and potential severe complications in future pregnan- 195
cies. In our case, the patient continued to receive RhIg ther-
apy, which could have been erroneously excluded if the
possibility of an existing anti-G rather than an anti-D
antibody was not considered. Our patient delivered a healthy
newborn at 39 weeks gestation who typed as A Rh(D) posi-
tive and had a negative DAT. No therapeutic interventions
were required.
 Keywords: anti-D, anti-C, anti-G, Rh immune globulin,
hemolytic disease of the newborn
1. Judd J. Summary of RAP Session Serologic Controversies (Two G, not CD).
AABB News. December 2000.
2. Shirey R, Mirabella D, Lumadue J, et al. Differentiation of anti-D, -C, and-G:
clinical relevance in alloimmunized pregnancies. Immunohematology.
1997;37:493.
3. Palfi M, Gunnarsson C. The frequency of anti-C and anti-G in the absence of
anti-D in alloimmunized pregnancies. Transfusion Med. 2001;11:207-210.
4. Cash K, Brown T, Strupp A, et al. Anti-G in a pregnant patient. Transfusion.
1999;39:531-533.
5. Hadley AG, Poole GD, Poole J, et al. Hemolytic disease of the newborn due to
anti-G. Vox Sang. 1996;71:108-112.
6. Yesus YW, Akhter JE. Hemolytic disease of the newborn due to anti-C and anti-
G masquerading as anti-D. Am J Clin Pathol. 1985;84:769-72.

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