Laboratory Monitoring of Mother, Fetus, and Newborn in Hemolytic Disease of Fetus and Newborn

Abstract

Background: Laboratory monitoring of mother, fetus, and newborn in hemolytic disease of fetus and newborn (HDFN) aims to guide clinicians and the immunized women to focus on the most serious problems of alloimmunization and thus minimize the consequences of HDFN in general and of anti-D in particular. Here, we present the current approach of laboratory screening and testing for prevention and monitoring of HDFN at the Copenhagen University Hospital in Denmark.

Summary: All pregnant women are typed and screened in the 1st trimester. This serves to identify the RhD-negative pregnant women who at gestational age (GA) of 25 weeks are offered a second screen test and a non-invasive fetal RhD prediction. At GA 29 weeks, and again after delivery, non-immunized RhD-negative women carrying an RhD-positive fetus are offered Rh immunoglobulin. If the 1st trimester screen reveals an alloantibody, antenatal investigation is initiated. This also includes RhD-positive women with alloantibodies. Specificity and titer are determined, the fetal phenotype is predicted by non-invasive genotyping based on cell-free DNA (RhD, K, Rhc, RhC, RhE, ABO), and serial monitoring of titer commences. Based on titers and specificity, monitoring with serial peak systolic velocity measurements in the fetal middle cerebral artery to detect anemia will take place. Intrauterine transfusion is given when fetal anemia is suspected. Monitoring of the newborn by titer and survival of fetal red blood cells by flow cytometry will help predict the length of the recovery of the newborn.

Keywords: Alloimmunization; Cell-free DNA; Hemolytic disease of fetus and newborn; Middle cerebral artery, Peak systolic velocity; Next-generation sequencing.

Fig. 1

The flow chart illustrates the use of individual components of the laboratory HDFN monitoring of all pregnant women. RhD-positive and RhD-negative women are screened for irregular antibodies against RBC antigens in the 1st trimester. Antibodies to ABO blood group antigens are only included in the examination if supplementary information gives an indication to do so. RhD-positive women who test negative at this first antibody screening are not examined later. All RhD-negative women are re-tested for antibodies at GA 25 weeks, and the antenatal RHD screening based on cffDNA is performed. RHD screening showing an RHD-positive fetus leads to the administration of Rh prophylaxis at GA 29 weeks and subsequently also the postnatal prophylaxis. Immunized RhD-negative women are screened for antibodies again at GA 32 weeks. At GA 25 weeks and GA 32 weeks, RhD-positive women with irregular antibodies detected in the first trimester are screened. Alternative timing of examination for both RhD-negative and positive women is followed if the clinician decides so. * Shows a potential trigger for conducting antenatal antigen prediction. The specific criteria for antenatal antigen prediction are: C, c, K, or D at titer ≥1; E at titer >1; A at titer ≥512; B at titer ≥256. For blood groups A and B, also HDFN due to anti-A or anti-B in a previous pregnancy gives an indication for antigen prediction. ** Shows a potential trigger for referral to the fetal medicine center. The specific criteria for referral are: anti-D, -C, -E, -e, -Cw, -Kpa, -Kpb,- k, -Jka, -Jkb, -Fya, -Fyb,-S, -s, -Wra, -M, -P1, -Lua, -Lub titer >16, and anti-K, -c titer ≥1. *** Designates postnatal monitoring of the newborn with titer, serological antigen detection, and flow cytometric quantification of fetal and donor RBCs. A black box designates an analysis, a grey box designates a result, and a white box designates Rh prophylaxis.

Fig. 2

Serial determinations of parameters for the first 72 days after the birth of the newborn. On day 19, less than 0.6 g/dL (0.4 mmol/L) of the total Hb 7.1 g/dL (4.3 mmol/L) was from endogenous E-positive RBCs. Only after 47 days, at a titer of anti-E below 4, did the E-positive RBCs survive and total Hb started to increase in parallel. Reticulocytes transiently decreased as a response to transfusions, and decreased again after day 61, when E-positive RBCs were being normalized. The newborn parameters were: anti-E titer determined by 2-step dilutions and analysis in CAT; total Hb (g/dL) from donor RBCs and newborn RBCs measured in a hematology analyzer; E-positive Hb was calculated as the E-positive RBC fraction of total RBCs measured by FC multiplied with the total Hb; reticulocytes were measured in a hematology analyzer. Informed consent was obtained from the parents of the infant. The study followed the guidelines of the institutional review board of Copenhagen University Hospital.