What is the role of serologic testing in the workup of hemolytic disease of the newborn (HDN)?

Updated: Dec 28, 2017
  • Author: Sameer Wagle, MBBS, MD; Chief Editor: Muhammad Aslam, MD  more...
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Serologic test findings include the following:

  • Indirect Coombs test and direct antibody test results are positive in the mother and affected newborn. Unlike Rh alloimmunization, direct antibody test results are positive in only 20-40% of infants with ABO incompatibility. [37] In a recent study, [38] positive direct antibody test findings have a positive predictive value of only 23% and a sensitivity of only 86% in predicting significant hemolysis and need for phototherapy, unless the findings are strongly positive (4+). This is because fetal RBCs have less surface expression of type-specific antigen compared with adult cells. A prospective study has shown that the titers of maternal immunoglobulin G (IgG) anti-A or anti-B may be more helpful in predicting severe hemolysis and hyperbilirubinemia. The sensitivity and specificity of IgG titers of 512 or higher in predicting need for invasive intervention was 90% and 73%, respectively. [39]  In a retrospective study (2005-2014) in a regional Belgian population, routine testing of maternal serum for relevant erythrocyte antibodies showed that in mother with positive antibodies, significant hyperbilirubinemia was noted more often if cord direct antiglobulin test (DAT) was positive (15% vs 2.6%). [40]  Significant hyperbilirubinemia was was noted in 7%, 11%, and 27%, respectively, in those with clinically relevant erythrocyte antibodies, anti-A and anti-B.  

  • Although the indirect Coombs test result (neonate's serum with adult A or B RBCs) is more commonly positive in neonates with ABO incompatibility, it also has poor predictive value for hemolysis. This is because of the differences in binding of IgG subtypes to the Fc receptor of phagocytic cells and, in turn, in their ability to cause hemolysis.

  • IgG2 is more commonly found in maternal serum but has weak lytic activity, which leads to the observation of little or no hemolysis with a positive direct antibody test result. On the other hand, significant hemolysis is associated with a negative direct antibody test result when IgG1 and IgG3 are predominant antibodies, which are in low concentration but have strong lytic activity, crossing to neonatal circulation.

  • In newborns with hemolytic disease due to anti-c or anti-C antibodies, direct antibody test results may be negative, and the diagnosis is established after indirect Coombs testing.

  • More recent studies on antibody characteristics have shown that lower core fucosylation of Rh-D antibodies while glycosylation and sialylation of anti-c antibodies significantly correlated with disease severity and fetal/neonatal disease. [41]

Obtain paternal molecular RhD zygosity testing. In addition, fetal RhD genotyping via cell free fetal DNA testing has become increasingly accurate in detecting fetal RhD allosensitization. [3]

Table. Comparison of Rh and ABO Incompatibility (Open Table in a new window)




Clinical aspects

First born



Later pregnancies

More severe

No increased severity




Severe anemia




Moderate to severe, frequent


Late anemia



Laboratory findings

Direct antibody test


Weakly positive

Indirect Coombs test


Usually positive




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