Case 16

Published on 18/02/2015 by admin

Filed under Allergy and Immunology

Last modified 22/04/2025

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CASE 16

DF is a 25-year-old woman, pregnant for the third time. She is known to have blood group A, Rh negative (Rh−) red cells. Her husband is also type A but Rh positive (Rh+). Their first-born child, a boy, was healthy but during the second pregnancy DF was noted to have an indirect Coombs’ titer of 1:32 in her serum. This fetus was followed closely, and a healthy baby girl was induced (vaginal delivery) at 36 weeks of gestation. DF received RhoGam after this delivery. It is now 3 years later and DF is pregnant again. You are monitoring her indirect Coombs’ antibody titer and find it to be 1:32 at 12 weeks and 1:48 at 18 weeks. Amniotic fluid was obtained beginning at 22 weeks of gestation and every 2 weeks thereafter. The amniotic fluid was shown to have increasing amounts of bilirubin (a pigment derived from a breakdown of heme), suggesting hemolysis of fetal red cells. At 28 weeks of gestation, a blood sample obtained from the umbilical vein revealed a hematocrit of 6.2% (normal 45%), confirming profound anemia in the fetus.

QUESTIONS FOR GROUP DISCUSSION

1. The significant point in this case is that the mother is Rh− and the father is Rh+. What is the Rh antigen? What percent of the population is Rh+? What is the potential problem for a fetus whose mother is Rh− and the father is Rh+?

2. Why is the risk of hemolytic disease of the newborn lower during a first pregnancy than for subsequent pregnancies? Include in your discussion how the mother could become sensitized to the Rh antigen during a first pregnancy? At the time of delivery?

3. Explain how maternal anti-Rh antibodies serve as a stimulus for complement-mediated hemolysis of fetal red blood cells. Describe the possible mechanism by which red blood cells would be destroyed after binding of IgG.

4. RhoGam is given to Rh− mothers if there is evidence of bleeding during pregnancy and especially at birth. What is RhoGam?

5. Explain (a) the “older” idea of how RhoGam prevents sensitization of the mother during placental bleeding or during the birthing process and (b) conventional wisdom as to how RhoGam works.

6. Define hematocrit and describe how it is determined. Under what circumstances would the fetal hematocrit continue to drop despite the administration of red blood cells into the umbilical vein?

7. Explain why the fetus was transfused with Rh− packed red blood cells when we already know that the fetus’s red blood cells are Rh+.

8. Explain how ABO incompatibility could reduce the likelihood of an Rh− mother being sensitized to the Rh antigen when carrying a child.

9. What test can be used to determine if the mother has circulating anti-Rh antibodies? At what antibody titer is the fetus considered to be at risk for hemolytic disease?

10. Explain how the serum of an Rh− mother who is pregnant with an Rh+ child gives a negative indirect Coombs’ test but agglutinates Rh+ cell in saline (see Case 15).

RECOMMENDED APPROACH

Implications/Analysis of Family History

The Rh antigen incompatibility between DF and her husband is of significance here because she is pregnant. The Rh antigen, so named because it was first identified on rhesus monkey erythrocytes (red blood cells), is expressed by about 85% of humans. When an Rh− individual becomes exposed (sensitized) to red blood cells expressing the Rh antigens, an immune response is generated because this is a foreign antigen. IgM and IgG anti-Rh antibodies are formed. This is of concern when an Rh− female is pregnant because IgG antibodies cross the placenta (see Etiology).

Differences in red blood cell antigens are more common in the ABO classification. ABO refers to the blood group cell surface glycolipids (antigens) used to classify red blood cells into four major groups: (1) red blood cells with A glycolipids, (2) with B glycolipids, (3) with both A and B glycolipids, and (4) red blood cells that do not express these antigens. Individuals are classed as A, B, or AB positive. Individuals who do not express either the A or B antigens are designated as having “O” blood type. In this case both DF and her husband were “A” positive, and so there was no incompatibility.

Implications/Analysis of Clinical History

This couple’s first child was healthy, and there is no indication of any problem associated with the Rh incompatibility. During DF’s second pregnancy, careful monitoring of antibody titers for anti-Rh antibodies indicated that DF had been sensitized to red blood cells expressing the Rh antigen and had generated anti-Rh IgG antibodies, but her antibody titer was relatively low. Antibody titer refers to the highest dilution of the serum that still induces agglutination of particles coated with the Rh antigen (see Implications/Analysis of Laboratory Investigation). After this delivery, DF was given RhoGam, an IgG anti-Rh antibody preparation. The mechanism by which it mediates its effects is not clear (see Etiology).

Implications/Analysis of Laboratory Investigation

Direct Coombs’ Test

The direct Coombs’ test is used to detect the presence of maternal anti-Rh IgG antibodies that have bound to the fetal red blood cells (see Case 15). The subsequent addition of anti-Fcγ antibodies, generated in another species, manifests in agglutination when (maternal) IgG antibodies are bound to the fetal red blood cells. This test is used when testing newborns for the presence of maternal IgG antibodies bound directly to the Rh antigen on their red blood cells.

Indirect Coombs’ Test

In contrast, in the indirect Coombs’ test we are testing for the presence of circulating anti-Rh IgG antibodies in serum (see Case 15). Therefore, an additional step is required. Red blood cells may be obtained from any source, the criteria (in this case) being that they express the Rh antigen. The mother’s serum is then allowed to react with these (allo or xeno) red blood cells, which will bind anti-Rh antibodies (if) present in the mother’s serum. Thereafter, the test is the same as that described for the direct Coombs’ test. Anti-IgG antibodies are added and the sample is considered positive for anti-Rh antibodies if agglutination manifests (see Case 15).

Bilirubin

The amniotic fluid was monitored for the presence of bilirubin, which would indicate that fetal red blood cells are being lysed. Bilirubin, a pigment derived from the breakdown of heme (following phagocytosis of red blood cells) is released into plasma (or in this case into the amniotic fluid) where it can be detected. Red blood cells are packed with hemoglobin, a tetrameric protein synthesized by nucleated precursors (while in the bone marrow). A heme prosthetic group, to which an iron atom is incorporated, is covalently attached to each hemoglobin subunit. Hemoglobin is able to carry oxygen to the tissues because oxygen binds reversibly to the iron. The presence of bilirubin in the amniotic fluid, therefore, is an indication that fetal red blood cells are being destroyed.

Hematocrit

Under these conditions there is concern that the fetus might be anemic and so a blood sample was obtained from the umbilical vein to determine the volume of packed red blood cells in a given unit of blood (hematocrit). A hematocrit of 6.2% (normal 45%) indicated profound anemia in the fetus.

Additional Laboratory Tests

Therapy was initiated (see Therapy) and the hematocrit monitored at 31, 34, and 35 weeks of gestation. The fetus was examined at weekly intervals for the appearance of hydrops (fetal subcutaneous tissue edema), caused by altered oncotic pressure differences (serum vs. interstitial fluid) after hemolysis, with consistently negative results. To determine when the fetus was sufficiently mature to sustain extrauterine life without difficulty, measures of surfactant were taken regularly. Infants born prematurely may have a risk of respiratory distress syndrome if they lack surfactant in alveoli. In the absence of surfactant the alveoli collapse during expiration.

DIAGNOSIS

The fetus was determined to have profound anemia.

THERAPY

Because the fetus was profoundly anemic, 85 mL of type O, Rh− packed red blood cells were transfused into the umbilical vein. At 31 weeks of gestation another sample of blood was obtained from the umbilical vein and tests indicated that the hematocrit was only 19%, indicating that the fetus’s red blood cells were still being destroyed. The fetus was transfused with an additional 65 mL of type O, Rh− packed red blood cells. At 34 weeks of gestation the hematocrit of a blood sample from the umbilical vein was 22%, resulting in an order for a further 70 mL of type O Rh− packed red blood cells to be transfused into the umbilical vein. At 35 weeks of gestation all measures indicated that the fetus was sufficiently mature (especially pulmonary/cardiac) to sustain extrauterine life without difficulty. Accordingly, labor was induced and a normal female child was delivered. The hematocrit in the umbilical vein blood at this time was 30%. The infant did well thereafter, and no further therapeutic measures were necessary.

HEMOLYTIC DISEASE OF THE FETUS

The rhesus incompatibility between an Rh− mother and an Rh+ offspring (the latter inherited Rh+ from the father) results in the production of anti-Rh antibodies when the mother is exposed to Rh+ red blood cells. This generally happens during birth in the first pregnancy but may occur before that if there is placental bleeding. The anti-Rh antibodies, so formed, bind any Rh+ red blood cells and this can cause red blood cell hemolysis in subsequent fetuses when IgG antibodies cross the placenta.

We generally assume (if this cannot be measured ahead of time) that every pregnant woman is Rh− and carrying an Rh+ child. Under these circumstances RhoGam is given as a precaution for any bleeding during pregnancy, and especially at birth. RhoGam is given to prevent sensitization and production of anti-Rh antibodies by the mother. This is not necessary if we know the mother is Rh+!

Mode of Action: RhoGam

RhoGam consists of an IgG anti-Rh antibody preparation. The mechanism by which it mediates its effects is not clear. It was previously believed that RhoGam “mopped up” or masked Rh antigen so the immune system never saw this antigen as a foreign determinant. In fact the amount of RhoGam given is not probably adequate for this to be the case. Conventional wisdom suggests instead that the immune complex formed (Rh:anti-Rh) is preferentially tolerogenic and induces regulatory cells of some kind.

Destruction of Red Blood Cells

The formation of antigen-antibody complexes on the surface of red blood cells would suggest that their destruction is the result of complement activation. Recall, however, that activation of complement requires the strategic location of two IgG antibodies so that C1q can simultaneously bind two Fc regions, a requirement for complement activation. It has been suggested that Rh antigens are so sparsely scattered at the red cell surface that IgG molecules bound to the Rh antigens are too far apart to fix C1q for complement-mediated hemolysis to occur. In consequence, it is thought that red blood cells (coated with anti-Rh IgG antibodies) adhere tightly to Fcγ receptors on macrophages present in the red pulp of the spleen and Kupffer cells of the liver. This results in FcR opsonin-mediated phagocytosis and destruction. Consistent with this hypothesis, most IgG antibody to the Rh antigen is of the IgG3 or IgG1 subclass, the IgG subclasses that bind most tightly to the high-affinity Fcγ receptor (CD64) present on macrophages and neutrophils.

Anti-Rh Antibody Titers

As a rough “rule of thumb” the amount of RhoGam we give passively (and safely, even if there is a Rh+ fetus present) is estimated to result in a maternal titer in the indirect Coombs’ test of less than 1:4. Thus, we acknowledge that such a titer of Rh antibody cannot cause significant hemolysis in the fetus. If the maternal titer is greater than 1:4, she has been alloimmunized by her fetus and the fetus is in need of careful monitoring. Note that the transfusion must be of Rh− red blood cells!

Effect of IgM Anti-Rh Antibodies on a Coombs’ test

Consider a scenario in which the serum of an Rh− woman, who is pregnant, gives a negative indirect Coombs’ test but agglutinates Rh+ cells suspended in saline. The most likely explanation for this phenomenon is that she has IgM anti-Rh antibodies that, being pentameric, are very effective in agglutinating Rh+ cells in saline, unlike IgG anti-Rh antibodies. However, because IgM antibodies do not cross the placenta they are unlikely to cause a problem with the pregnancy. It would be wise to monitor her anti-Rh antibodies to ensure she does not (later) develop a positive indirect Coombs’ test, signifying the presence of IgG antibodies.

Effect of ABO Incompatibility on Rh Alloimmunization

Note that ABO incompatibility between mother and father can itself modulate immunization to Rh determinants. Why? In the presence of ABO incompatibility, the mother would already have preexisting anti-A or anti-B IgM antibodies (isohemagglutinins). Therefore, when (and if) fetal blood enters the maternal circulation, IgM immune complexes would activate complement; fetal red blood cells would be quickly hemolyzed. This rapid destruction/elimination of the fetal red blood cells would decrease the likelihood of Rh alloimmunization of the mother.

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