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17. Rhesus-Negative Mother
17.1 Introduction
Hemolytic disease of the newborn (HDN) secondary to rhesus alloimmunization was once a major contributor to perinatal morbidity and mortality. However, with the advent of anti-D immunoprophylaxis, the disease prevalence and the associated morbidity and mortality are markedly reduced with approximately 1–6 cases occurring every 1000 live births [1]. However, the disease is still a major problem in developing countries, including India with an estimated 56,700 cases of Rh-HDN occurring annually [2].
The incidence of Rh incompatibility varies by race and ethnicity. Approximately 15% of whites are Rh negative, compared to only 5–8% of African Americans and 1–2% of Asians [3]. Around 5% of the Indian population is Rh negative.
17.2 Pathophysiology
The RhD polypeptide is an integral membrane protein exclusively expressed on erythrocytes and identified as early as 38 days after conception [4]. With advancing gestational age, the volume and frequency of fetomaternal hemorrhage (FMH) increase with quoted rates being 3%, 12%, and 46% of women in each of the three successive trimesters [5]. The volume required to cause alloimmunization depends upon the immunogenicity of the Rh-positive erythrocytes and the immune responsiveness of the mother.
After fetomaternal hemorrhage in a Rh-negative mother, the initial response to D antigen exposure is slow and produces IgM anti-D. On subsequent antigenic exposure, there is rapid production of IgG anti-D antibodies which cross the placenta and coat D-positive fetal erythrocytes. Anti-D IgG is a non-agglutinating antibody which does not bind complement; hence, there is no intravascular hemolysis, but sequestration and subsequent destruction of antibody-coated red cells in the fetal liver and spleen is the mechanism of fetal anemia. Mild-to-moderate hemolysis is manifested as increased amniotic fluid indirect bilirubin levels. Severe hemolysis leads to increased red blood cell production by the spleen and liver of the fetus leading to portal hypertension and placental edema and eventually fetal ascites. Hepatomegaly, increased placental thickness, and polyhydramnios usually precede the development of fetal heart failure. Further liver damage decreases albumin production leading to the development of hydrops fetalis [1]. Coexistent ABO incompatibility causes rapid clearance of incompatible red cells and, hence, reduces the overall exposure to D antigen and, therefore, decreased risk of alloimunization.
17.3 Tests for Fetomaternal Hemorrhage
Kleihauer-Betke acid-elution test is the most widely used test for quantifying FMH [6]. Fetal RBCs mainly contain fetal hemoglobin (HbF), which is resistant to acid elution, while adult hemoglobin is acid-sensitive. The test is performed on the mother’s blood; the blood undergoes acid elution and staining. The acid-resistant fetal cell stains red; maternal cells stain pink. However, its limitations include (1) cases of maternal hemoglobinopathies where maternal red cells carry excess fetal hemoglobin and (2) cases nearing term, when fetus has already started to produce hemoglobin A [7].
Flow cytometry is an alternative technique for quantifying the size of FMH [8]. Its results are more accurate and more reproducible compared to Kleihauer test, and since it detects RhD-positive cells, it is particularly useful in patients with high HbF levels. It is used when a Kleihauer screening test indicates a large FMH which requires accurate quantification and follow-up. The rosetting technique is another serological method used to quantify FMH of RhD-positive red cells.
A 300 μg intramuscular dose of anti-D immunoglobulin is sufficient for 15 mL of fetal red cells or 30 mL of fetal whole blood. The additional dose of anti-D should be calculated on the basis of extra 20 μg for each mL of fetal cells present. A maternal sample should be collected 72 h after the intramuscular administration of anti-D (48 h of intravenous anti-D) to assess the removal of fetal cells following a large fetomaternal hemorrhage. More anti-D may be necessary if fetal cells remain.
No more than five units of rhesus immune globulin should be administered by the intramuscular route in one 24-h period. If using an intravenous preparation, two ampoules (600 μg) may be given every 8 h [1].
17.4 Management of Sensitized Antenatal Mother
A titer of more than 1:4 is considered sensitized. Once Rh antibodies are detected, the next step is to determine the concentration of antibodies and the likelihood of HDFN. It is important to find out if the antibodies are immune or passive, and history of any prior anti-D injection within last 12 weeks should be elicited.
17.4.1 Distinguishing Between Passive and Immune Anti-D
The concentration of passive anti-D Ig in maternal samples post-prophylaxis rarely exceeds 1:4 following a dose of 300 μg. Passive anti-D Ig can be detected in the blood within minutes after intramuscular injection, reaches peak concentration in 3–7 days, and can be detected for more than 12 weeks by an indirect antiglobulin test (IAT). Immune anti-D on the other hand becomes detectable only after 4 weeks of exposure to D-positive cells and reaches a peak concentration after 6–8 weeks, if there is no further exposure [9].
On serial monitoring, if the anti-D level is falling, it is probably passive, whereas if it is steady or rising, it is probably immune. Prophylactic anti-D should continue unless it is established that the anti-D is immune.
A critical titer is defined as the titer significantly associated with development of fetal hydrops. This titer varies with institutions based on clinical correlation with hemolytic disease of the newborn and is between 8 and 32 in most centers. In Europe and the United Kingdom, the concentration of anti-D is measured in IU/mL. A threshold value of 15 IU/mL has been recommended for invasive testing as values below this are usually associated with only mild hemolytic disease of the newborn [10].
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