A Curious Case of Anti-D Antibody Titer

Jennifer G. Hensley, CNM, WHNP, EdD; Katherine P. Coughlin, CNM, MSN; Laura L. Klein, MD

J Midwifery Womens Health. 2009;54(6):497-502.

Laboratory Testing

There is no laboratory test to distinguish between active (alloimmunization) or passive anti-D antibody (RhIG administration) production. One or the other is assumed by the strength of RBC reaction to agglutinins that test for anti-D antibody.^[1] A titer from passive RhIG administration may reach 1:4; a titer ≥ 1:8 requires further investigation for RhD alloimmunization.^[1] Measurement of the amount of anti-D antibody in the plasma is variable between and within laboratories. Variability in performing and reporting a titer result is caused by differences in available reagents, shelf life of the reagents, equipment used, and operator competence. For these reasons, 1) the same laboratory should be used to run the titers, and 2) a four-fold increase, or 2-tube dilutional change, becomes necessary to qualify as a true increase in the amount of antibody production that corresponds with a risk of hemolytic disease of the fetus and newborn. For example, a titer increase from 1:8 to 1:16 is not significant, but an increase from 1:8 to 1:32 is significant.^[1] Each laboratory sets its own "critical" titer for anti-D antibody that requires follow-up; this is the titer at which fetal hydrops is likely to occur.^[1] The critical titer could be 1:8, 1:16, or 1:32. The clinician will need to know the critical titer at their laboratory to effectively interpret high titer results.

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Table 1. Definitions for Technical Terms Related to Rh Alloimmunization
RhIG	Passive anti-D immune globulin administered to prevent anti-D antibody formation in RhD-negative women who carry and are exposed to RhD-positive fetal RBCs; once called Rh "sensitization," this is now known as RhD alloimmunization Manufactured by four companies in the United States, RhoGAM was the first product on the market and is the one most often referenced
Antibody titration studies¹	Semiquantitative method of determining the amount of anti-D antibody in the maternal plasma Serial two-fold dilutions of serum are prepared and tested for antibody activity and the highest dilution that produces a1+ agglutination is reported as the titer (e.g., 1:2, 1:4, 1:8, 1:16, 1:32, etc.)
DAT or direct Coomb test¹	A test on fetal or newborn RBCs to determine if the RBCs have been coated with maternal anti-D antibody The test itself is not diagnostic
IAGT or indirect Coomb test¹	Maternal RhD-negative RBCs are incubated in serum, washed to remove free antibody, and then exposed to anti-D Hemagglutination would be positive for maternal cells that have been bound with anti-D Also known as the "antibody screen" that is drawn before the administration of RhIG to RhD-negative women.
Kleihauer-Betke acid elution tes¹	Fetal hemoglobin resists release (or elution) from its RBC under acid conditions, whereas adult hemoglobin is released. The mother's blood is drawn and a sample smear prepared and exposed to an acid buffer. Fetal RBCs remain intact, and the volume is calculated from the smear. The percent of fetal RBCs is multiplied by 50 to determine the mL of fetal RBCs in maternal circulation This test can be inaccurate and imprecise, especially when determining the exact amount of RhIG to administer to an RhD-negative woman, following a large FMH. The practitioner should err on the side of safety and administer an additional 300 mcg of RhIG
Rosette test; a qualitative test for identification of fetal RBCs in maternal blood¹	Tests for the presence of fetal RhD-positive cells in the blood of an RhD-negative mother Blood from the mother is collected and anti-D is added to coat the RhD-positive fetal RBCs. RhD-positive reagent is subsequently added. The RhD-positive fetal RBCs, coated with anti-D, react to form a "rosette" around the antibody-coated cells The absence of a rosette is negative The presence of a rosette is positive. A positive rosette is evidence of a FMH of approximately 10 mL; a quantitative test, such as the Kleihauer-Betke test, should be performed
Sensibilization³	Confirmed anti-D antibody in the plasma of an RhD-negative woman who reverts to a negative antibody status (e.g., 1:16 → undetectable antibody). Memory of the RhD-positive antigen is retained on B cells. During a subsequent pregnancy with an RhD-positive fetus, the woman will produce anti-D antibodies against the antigen that will again become detectable in the plasma or serum This woman is really alloimmunized
Sensitization or alloimmunization³	Production of anti-D antibodies by an RhD-negative mother that are capable of crossing the placenta and hemolyzing RhD-positive fetal RBCs, causing HDFN The woman's anti-D antibody titer remains detectable

DAT = direct agglutination test; FMH = fetal-maternal hemorrhage; HDFN = hemolytic disease of the fetus and newborn; IAGT = indirect agglutination test; RBCs = red blood cells; RhIG = Rh_o immune globulin, anti-D immune globulin.
Sources: Roback et al.¹ and Moise.³

Table 2. When to Administer Rh _o Immune Globulin
Indication	Dose
After an ectopic pregnancy	Mini RhIG
After a first-trimester loss	Mini RhIG
After a first-trimester termination	Mini RhIG
After CVS	Mini RhIG
Threatened abortion	Mini RhIG
Hydatidiform mole	Full-dose RhIG
After amniocentesis	Full-dose RhIG
After cordocentesis	Full-dose RhIG
Prophylactically at 28 weeks' gestation	Full-dose RhIG
Second- or third-trimester bleeding	Dose dependent upon no. of fetal RBCs in maternal circulation as determined by the Kleihauer-Betke test
External cephalic version	Full-dose RhIG
Suspected abruption placenta	Dose dependent upon no. of fetal RBCs in maternal circulation as determined by the Kleihauer-Betke test
Abdominal trauma	Dose dependent upon no. of fetal RBCs in maternal circulation as determined by the Kleihauer-Betke test
Intrauterine fetal demise	Dose would depend upon cause; dose may be dependent upon number of fetal RBCs in maternal circulation as determined by the Kleihauer-Betke test
Within 72 hrs postpartum after delivery of an RhD-positive fetus	Dose dependent upon no. of fetal RBCs in maternal circulation as determined by the Kleihauer-Betke test

CVS = chorionic villus sampling; RhIG = Rh_o immune globulin, anti-D immune globulin; RBC = red blood cell.
Source: American College of Obstetricians and Gynecologists.²

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A Curious Case of Anti-D Antibody Titer

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