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Hemolytic disease of the fetus and newborn is reliably prevented by proper management, based on antenatal D typing and screening for red blood cell (RBC) antibodies. Many hospital laboratories do not determine the RHD genotype of pregnant women with a serologic weak D phenotype, because these women are often managed as D–.1 However, Rh immune globulin (RhIG) and provision of D– RBC units are unnecessary if D+ RBCs can safely be transfused. An Interorganizational Work Group on RHD Genotyping recommended in 20152 to phase-in RHD genotyping for patients with a serologic weak D phenotype. The same authors3 specified their recommendation in 2020 to phase-out the reporting of a “serologic weak D phenotype” and resolve all weak D types with RHD genotyping.
We report a pregnant woman with anti-U and a serologic weak D phenotype. The clinical workup in this case illustrated the importance of molecular analysis of serologic weak D phenotypes early in the pregnancy to preserve rare D– RBC units and to eliminate the unnecessary administration of RhIG.
Case Report and Results
A 23-year-old African American woman (gravida 2, para 1) presented for childbirth. The woman had no history of blood transfusion. Testing of her blood sample showed her RBCs to be group B with a serologic weak D phenotype; anti-U was identified by the antibody screening process (Table 1). Without any molecular information for her RHD genotype, the woman was initially considered to be managed as D–. We decided to obtain 3 U– RBC units; however, only 1 was D–.
Clinical laboratory results for mother and neonate
Test
Results (normal range)
Maternal
Transfusion medicine†
ABO group
B
RhD phenotype
Serologic weak D phenotype: 2 + reaction strength‡
RhCE phenotype
C–E–c+e+
Antibody screen
Anti-U
DAT
Negative
Red cell genotyping†
RHD allele
RHD*weak D type 4.0
RHD zygosity
Hemizygous
Hematology†
Hemoglobin, g/dL
Antepartum
9.8 (10.0–15.0)
Postpartum
8.9 (10.0–15.0)
Neonatal
Transfusion medicine§
ABO group
B
RhD phenotype
D+
RhCE phenotype
C–E–c+e+
DAT
Negative
Red cell genotyping§
RHD allele
RHD*weak D type 4.0 and normal RHD
RHD zygosity
Compound heterozygous
Hematology
Hemoglobin, g/dL
17.6 (14.0–24.0)
Unconjugated bilirubin, mg/dL
At birth
4.9 (<6)
4 hours after birth
5.5 (<6)
Reticulocyte count, %
4.46 (3.0–7.0)
At the end of the third trimester.
Tested by the conventional tube method at immediate spin (Anti-D Blend, oligoclonal antibody mixture with clone numbers BS232, BS221, and H41 11B7; Bio-Rad, Hercules, CA).
At birth.
DAT = direct antiglobulin test.
In the week before delivery, nucleotide sequencing of the RHD gene was performed on the mother and, later, on the neonate.4 Based on the three amino acid substitutions (Table 2), we concluded that the mother carried the RHD*weak D type 4.0 allele (Table 2) and was hemizygous for the RHD gene. The neonate was a compound heterozygote for the RHD gene with a RHD*weak D type 4.0 allele from the mother in trans to a normal RHD allele from the father. A total of 12 and 13 nucleotide changes were confirmed in the mother and neonate, respectively (Table 2).
Single nucleotide variants detected in the RHD gene
RHD genotype
Location
Nucleotide change*
dbSNP reference number
Protein residue change†
Mother‡
Neonate
Promoter
−368a>g
rs28710826
NA
g/g
a/g
Intron 2
336−76_−75−>insTGAA
rs112473736
NA
insTGAA/insTGAA
insTGAA/−
Intron 3
487−414a>g
rs28586271
NA
g/g
a/g
487−316t>g
rs28572396
NA
g/g
t/g
Exon 4
602C>G
rs1053355
Thr201Arg
G/G
C/G
Exon 5
667T>G
rs1053356
Phe223Val
G/G
T/G
Intron 5
801+219 t> g
rs28510210
NA
g/g
g/g
801+395g>a
rs145236797
NA
a/a
g/a
802−16c>t
rs201120463
NA
t/t
c/t
Exon 6
819G>A
rs150606530
Ala273Ala
A/A
G/A
Intron 6
939+295c>a
rs112222730
NA
c/c
c/a
Intron 7
1073+94g>a
rs533903485
NA
a/a
g/a
1073 + 311g> c
rs3118453
NA
c/c
c/c
Nucleotide substitutions are shown relative to the reference sequence (NG_007494.1). Nucleotide positions are defined using the first nucleotide of the coding sequence of NM_016124.4 isoform as nucleotide position 1. The uppercase nucleotides are located in the coding sequence, and the lowercase nucleotides are located in the non-coding sequence of the RHD gene.
Relative to the National Center for Biotechnology Information (NCBI) Reference Sequence NP_057208.2.
The nucleotide sequence of the RHD*weak D type 4.0 allele comprising 8572 base pairs, detected in the mother hemizygous for one RHD gene, has been deposited in GenBank as accession number MT900842.
dbSNP = Single Nucleotide Polymorphism Database; NA = not applicable.
Zygosity testing for the RHD gene was done by a quantitative fluorescence–polymerase chain reaction (QF-PCR) assay.5 The mother was hemizygous (one copy) and the neonate was homozygous (two copies) for the RHD gene. The QF-PCR is the preferred method for RHD zygosity testing in individuals of African descent, although it is known to have limitations in white individuals where a restriction fragment– length polymorphism (RFLP) assay may be the more reliable method.6–8
We still applied an RFLP assay that is designed to detect the standard downstream Rhesus box, indicative of the presence of an RHD gene (i.e., lack of the RHD deletion).7 However, the mother who carried an RHD gene tested negative (seemingly no copies) in this RFLP assay, and the neonate who carried two copies of the RHD gene tested hemizygous for the RHD gene (seemingly only one copy). These discrepancies are explained by variations in the downstream Rhesus box of individuals of African ancestry and are a known limitation of the RFLP assay in these individuals.6,8,9
The mother, with an unexplained hemoglobin (Hb) concentration of 9.8 g/dL prepartum, had an uneventful vaginal delivery. Her Hb dropped by 0.9 g/dL, and she did not require transfusion (Table 1). The neonate’s blood sample typed as group B, D+ with normal clinical laboratory results (Table 1), and no treatment was required. The 2 U–, D+ RBC units were returned and used in the care of another pregnant woman with anti-U. The unnecessarily procured U–, D– RBC unit had to be frozen, however, with only 14 days of shelf-life remaining.
Discussion
The present clinical report exemplifies the advantage of RHD genotyping in expectant mothers to identify RHD alleles that allow the mothers to be safely treated as D+.3 The molecular analysis should be performed early in a pregnancy. This approach, which was missed at the first-time maternity visit in our patient, would have allowed for an efficient organization of RBC genotyping with or without antibody identification. Most hospitals would typically send samples to an immunohematology reference laboratory. In our case, while birth was imminent, the shipping and testing was accomplished within 5 days, including a weekend. The extra cost inflicted by this time constraint could surely have been avoided with better planning of the required tests during the pregnancy. Complex serologic and molecular testing in immunohematology are more prone to errors when performed under extreme time constraints and thus should be avoided.
The blood supply in transfusion service is often limited, especially for patients with the D– phenotype, and more so if antibodies to high-prevalence RBC antigens are also present.10 For the expectant mother in our study, a compatible donor with a U–, D– phenotype is extremely rare, representing <0.1 percent of the African American population.11 U–, D+ RBC units are also very rare, but there are five to ten times more donors if the D– restriction can be removed.
Supporting every patient who is D+ due to RHD*weak D type 4.0 with D– RBC units to prevent anti-D would be a burden and is discouraged.3,12 D– RBC transfusion and RhIG administration may be considered during pregnancy in an abundance of caution, although several health care systems are considering moving to an exclusively D+ transfusion management policy.3,13,14 Pregnant women with the RHD*weak D type 4.0 allele, who were never shown to produce an alloanti-D with adverse clinical outcome, could falsely be diagnosed of carrying an alloanti-D that is actually due to RhIG administration.13,14 This passively acquired anti-D can mislead the results of compatibility testing, when RBC units are crossmatched in preparation for delivery. Pitfalls of mistaking passive anti-D for active immunization can be avoided by obtaining a history and performing an anti-D titer.15 In summary, we decided to treat the current patient as D+ for transfusion purposes and recommended against RhIG administration during pregnancy and after the birth of the D+ baby boy.
Studies on cost and financial implications explored the economic aspect of RHD genotyping for pregnant women with a weak D phenotype.16,17 If the personal health information is properly maintained and shared, particularly in highly developed countries like the United States, RHD genotyping would add only a one-time testing cost for each pregnant woman with a weak D phenotype, while providing a rationale for the transfusion strategy during the rest of a woman’s life.18 This strategy could prevent unnecessary costs and risks associated with RhIG administration and follow-up scheduling during the current and every subsequent pregnancy.3 The best time to resolve a serologic weak D phenotype with RHD genotyping is early in the first pregnancy.2