Perinatal Transfusion Medicine

Testing for RhD Type

At the first prenatal visit, usually at 12 weeks of gestational age, the maternal ABO/RhD type and antibody screen are performed. A challenging area in the laboratory has been properly identifying patients who have a variant D phenotype. These individuals have altered or weakened expression of their D antigen, which often requires a sensitive antiglobulin test or other method to detect, referred to as a serologic weak D phenotype. Depending on the methodology and/or reagents, these patients may test as D-negative, weak D, or D-positive. This leads to confusion about whether these patients should be considered as candidates for RhIg prophylaxis and transfused D-negative blood products.

The three broad categories of D variants include weak D, partial D, and the rare DEL phenotypes, which are most common among those of Asian ancestry and are not detected by conventional serologic typing methods. In particular, individuals with weak D and partial D are often determined to have a serologic weak D phenotype when tested in the transfusion service. Weak D is a quantitative polymorphism resulting in reduced expression of D antigen; whereas, people with partial D can make alloanti-D because of a qualitative polymorphism resulting in an altered D antigen epitope. The majority of individuals with serologic weak D phenotype of European ancestry are weak D types 1, 2, or 3, and will not form anti-D when exposed to D-positive cells, but the percentage varies depending on ethnic background. The remainder, including an estimated 10% of individuals serotyped as weak D, who are partial D variants, can potentially form anti-D and should be managed as if they were D-negative. In 2015, recommendation for RHD genotyping for pregnant women and females of childbearing potential with a serologic weak D phenotype, was endorsed by AABB, America’s Blood Centers, the American Red Cross, the Armed Forces Blood Program, the College of American Pathologists, and the College of Obstetricians and Gynecologists. Specifically, an algorithm was published for resolving serologic weak D phenotype test results by genotyping to determine candidacy for RhIg and RhD type for transfusions (see Fig. 50.1 ).

RhIg Prophylaxis

If a pregnant woman is D-negative and is not sensitized to the D antigen, or has a serologic weak D phenotype that should be managed as D-negative, then she should receive RhIg perinatally.

Antepartum and postpartum dosing is usually with 300 μg of RhIg (which neutralizes 30 mL of whole blood or 15 mL of D-positive RBCs), although some institutions prefer to give 50 μg (which neutralizes 5 mL of whole blood or 2.5 mL of D-positive RBCs) at ≤12 weeks of gestational age, particularly with abdominal trauma or threatened miscarriage, but there is a risk of inadvertent misadministration of the lower dose (see Chapter 40 ). The half-life of passive anti-D is 3–4 weeks and is detectable in over half of women who deliver less than 76 days after administration of RhIg.

RhIg is administered at:

• 28 weeks of gestational age (dose 300 μg);

• at delivery, if the neonate is D-positive, weak D-positive, or D-untested (minimum dose of 300 μg, further dosing determined by FMH testing); and

• after perinatal events associated with FMH, such as abortion, ectopic pregnancy, amniocentesis, chorionic villus sampling, external cephalic version, abdominal trauma, and antepartum hemorrhage (minimum dose of 300 μg, further dosing determined by FMH testing, if >20 weeks of gestational age).

After a perinatal event or delivery beyond 20 weeks of gestational age, when the fetal blood volume exceeds 30 mL, quantification of FMH is recommended. In all situations, RhIg should be administered within 72 hours of the event. If a dose is not administered during that time frame, then it should be administered as soon as possible, even up to 28 days after the event. While passive anti-D may be detectable up to 6 months after administration, very few women (less than 3%) will have demonstrable anti-D after 76–95 days of RhIg administration. Therefore, any woman with detectable anti-D beyond this timeframe may not have had adequate antepartum protection and should be considered suspicious for the development of immune anti-D.

Fetomaternal Hemorrhage Testing

The goal of FMH testing is to determine an adequate dose of RhIg to neutralize fetal D-positive cells in the maternal circulation; thus, preventing maternal alloimmunization to RhD antigen. A sample for FMH testing should preferably be obtained approximately 1 hour after delivery on all D-negative women who deliver a D-positive infant (or 1 hour after an event as described above). FMH testing can be performed by a screening test, which is typically the rosette test. If positive, quantification of FMH is usually performed by an acid elution test (Kleihauer–Betke test) or by flow cytometry. Other FMH detection methodologies are in use, such as gel agglutination and enzyme-linked antiglobulin test.

Rosette Test (Fetal RBC Screen [Qualitative])

The rosette test demonstrates the number of D-positive cells in a D-negative suspension using an anti-D reagent. The anti-D binds to D-positive fetal RBCs, and when indicator D-positive RBCs are added rosettes are formed. This method has an FMH detection limit of about 10 mL. For the test to be valid, fetal cells must be D-positive (not weak D or D unknown—result is false negative) and the maternal cells must be D-negative—result is false positive; in those situations, a test that detects fetal hemoglobin should be used, such as the Kleihauer–Betke or flow cytometry test.

Kleihauer–Betke Test (Fetal RBC Count [Quantitative])

The Kleihauer–Betke test has several limitations, including low sensitivity, poor reproducibility, and tendency to overestimate the FMH volume, yet it is the most commonly used test to quantify FMH in the United States. The Kleihauer–Betke test is performed on a maternal blood smear treated with acid and then stained, so the fetal RBCs remain red, and the maternal RBCs appear as ghosts. 2000 cells are counted, and the percentage of fetal RBCs is determined.

Flow Cytometry (Fetal RBC Count [Quantitative])

Flow cytometry techniques quantitate the amount of hemoglobin F or D-positive RBCs and are simpler, more precise, reliable, and thus may result in lower RhIg administration than the Kleihauer–Betke test. Flow cytometry techniques are not routinely available throughout the United States.