The Frequency of Severe Hemolytic Disease of the Fetus and Newborn Caused by Red Blood Cell Alloantibodies Undetected at the Antibodies Screening in the First Trimester: A Prospective Study

Abstract

Introduction

The aim of this study was to investigate the clinical consequences of anti-D and non-anti-D antibodies undetected at screening in the first trimester for hemolytic disease of the fetus and newborn (HDFN).

Methods

This prospective study (conducted from 2010 to 2024) included pregnant women with negative first-trimester antibody screening for red blood cell (RBC) antibodies but positive later in pregnancy for clinically significant antibodies anti-D and non-anti-D. We analyzed the incidence of antibodies, DAT of the newborn, and the treatment and outcomes of HDFN.

Results

In study period, 61,200 pregnant women were tested, of which 11,000 were RhD−. In 32 cases, anti-D antibodies were initially detected at the repeated screening (28th/34th week of pregnancy or to 24 h post-delivery). All women have given birth to D+ child, of which 50% had a positive DAT. Four of them had early jaundice. In two cases, blood transfusion was required due to anemia. The incidence of anti-D antibody undetected at first trimester was 29/10,000 in D− women. Anti-D antibody caused severe HDFN in 1.8/10,000. Non-anti-D antibodies have been detected on repeated screening in seven cases, anti-c (N = 5), anti-E (N = 1), anti-C (N = 1). All seven neonates were antigen positive, three of which had positive DAT due to anti-c antibodies. The neonates with positive DAT had early jaundice and needed phototherapy, but there were no cases of severe HDFN in this group.

Conclusion

First-trimester screening for RBC antibodies detected all cases of severe HDFN caused by non-anti-D antibodies out of 61,000 pregnant women tested.

Introduction

Maternal red blood cell (RBC) antibodies cross the placenta and can cause hemolytic disease of the fetus and newborn (HDFN). Examination for RBC antibodies with the indirect antiglobulin test (IAT) is performed in all pregnant women during the first trimester of pregnancy, since the presence of antibodies at the beginning of pregnancy carries the greatest risk for the development of HDFN. Because of fetal-maternal hemorrhage (FMH) during pregnancy, a negative screening test may become positive, and antibodies detected on the follow-up testing are considered significant if they cause HDFN [1]. One of the key advantages of conducting antibody screening during pregnancy is that it ensures the delivery to occur in a hospital equipped to perform exchange transfusions (ETs) if necessary and guarantees that the newborn will be examined by a pediatrician. These preventive measures are also important for timely treatment and reduce the risk of late detection of HDFN in the newborn. The results of previous research studies pointed out that anti-D and non-D antibodies that were undetected in the first trimester of pregnancy can cause severe HDFN [2–4]. It is important to note that in most of the studies, the criteria for severe HDFN were based on the treatment of fetuses and newborns with intrauterine transfusions (IUTs), ETs, and simple blood transfusion (BT) in the first week of life [1–4]. Given that the criteria for transfusion treatment of newborns have become more restrictive in recent years, we can expect changes in the incidence of severe HDFN presentation [5, 6]. Also, a greater stringency to phototherapy is practiced in jaundice of hemolytic origin as well as use of more effective phototherapy devices and protocols [5–8].

In addition, more sensitive methods for IAT were used in our center, making it possible to detect low-titer red cell antibodies in first-trimester screening, which might be missed by a less sensitive test [9]. In view of the above, we prospectively followed pregnant women with negative screening for red cell antibodies in the first trimester, but with antibodies detected on repeated screening in pregnancy or delivery, and we observed whether these antibodies caused severe HDFN cured by transfusion. We compared the results with a historical control.

Methods

The study was performed in the Department of Transfusion Medicine, University Hospital of Split, Croatia. Our Hospital is a regional institution for the Split-Dalmatia County responsible for overall pregnancy care, monitoring of RBC alloimmunized women, and the management of deliveries. This study was conducted in the period of 14 years (2010–2023.) and included pregnancies with clinically relevant RBC alloantibodies detected at repeated screening during pregnancy or delivery, in which antibody screening at first trimester was negative. We prospectively followed pregnancies and collected data that characterized severe HDFN, namely, specific antigen of newborn, direct antiglobulin test (DAT) of newborn, IUTs and postnatal ETs, simple BTs, and phototherapy.

Protocol for Testing Pregnant Women

In Croatia, blood group and antibody screening for all pregnancies is performed during the first trimester. Repeated antibody screening is recommended for RhD-positive women at the 34th week of gestation. For RhD-negative women, repeated antibody screening is advised at both 28th and 34th weeks of pregnancy, as well as immediately following delivery. Rh immunoprophylaxis is administered to RhD-negative women following miscarriage, pregnancy termination, chorionic villus sampling, amniocentesis, and delivery. Antenatal immunoprophylaxis is not provided at 28th to 30th week. Screening for RBC antibodies was performed by IAT in a fully automated system OrthoVision^® using polyspecific microcards with commercially prepared RBC (0.8% Surgescreen I/II/III). In the cases where antibodies that mediate HDFN were detected, i.e., clinically relevant alloantibodies, such as anti-D, -K, -k, -c, -C, -E, Fy, -S, -s, -Jk, pregnant women were tested more frequently until the delivery by antibody titer and ultrasonography. Ultrasound is performed for MCA peak systolic velocity (MCA-PSV). MCA-PSV normograms are available and values are grouped according to multiples of median (MOM). In a case where MCA-PSV has reached 1.5 MOM level, MCA-PSV is repeated after 1 week and if it remains at this level or rises, and significantly high antibody titer (titer anti-K ≥4, anti-c ≥8, and anti-D other alloantibodies ≥16), it is an indication for cordocentesis and IUT. If titer has increased, but MCA-PSV is less than 1.5 MOM, the patient is still managed with weekly MCA-PSV measurement. If antibody titer is high or rising at gestational age of 35, MCA-PSV measurement is omitted; in that case, amniocentesis is the method of choice to detect bilirubin levels in amniotic fluid and induce labor [10, 11].

Testing of Newborn and Therapy of HDFN

The immunohematology tests of the neonates born from alloimmunized mothers included ABO blood group, RhD antigen or other specific RBC antigens and DAT. Standard laboratory tests comprised: complete blood count, hemoglobin and hematocrit levels, reticulocyte count, serum levels of total, indirect and direct bilirubin [5]. In our hospital, neonatologists decide whether to perform ETs, top-up transfusions, or phototherapy based on the current recommendations. For patients with severe anemia (hematocrit <25 percent) and/or severe hyperbilirubinemia (i.e., bilirubin at or above the hour-specific threshold for ET as established in the American Academy of Pediatrics [AAP] guidelines), an ET is performed [7]. UpToDate criteria are used in our institution to guide decision when performing simple transfusions in cases of moderate to mild anemia and phototherapy treatable hyperbilirubinemia. For neonates ≥35 weeks of GA and clinical instability, the recommended thresholds are as follows: 0–7 days old, Hgb <11 g/dL (Hct <32 percent); ≥8 days old, Hgb <7 g/dL (Hct <21 percent). When neurotoxicity risk factors for bilirubin-induced neurologic injury, as outlined by AAP guidelines, are identified, decisions regarding initiation of phototherapy or escalation of care are based on the infant’s gestational age, total serum bilirubin at a given hour, and any underlying risk factors. If such risk factors are present, both the threshold for beginning phototherapy and the criteria for escalating care are reduced accordingly. This approach to jaundice treatment leaves a high margin of safety with respect to the bilirubin-associated neuronal injuries [6, 8]. The study was approved by the Ethics Committee of the University Hospital of Split.

Results

Based on statistical data from Croatia, 44% of mothers were first-time mothers, 34% were para 2, and 22% were para 3 or more. A history of miscarriage or abortion was documented in 20.3% of pregnant women [12]. In our hospital, 61,200 pregnant women were tested for blood group and antibodies screening between January 1, 2010, and December 31, 2023, of them 11,000 (18%) were RhD-negative. In 32 cases of 11,000 RhD-negative pregnant women, anti-D antibodies were undetected at first-trimester screening, but the immunization was discovered on the repeated screening (28th/34th week of pregnancy or post-delivery). In all pregnant women with anti-D antibodies detected during pregnancy and childbirth, it was confirmed based on history that women did not receive immunoprophylaxis. The incidence of RhD immunizations discovered during pregnancy is 29/10,000 RhD-negative women. Out of these 32 pregnancies, 11 were primiparous women. All have given birth to RhD-positive children, of which 50% (16 out of 32) had a positive DAT (shown in Table 1). Table 2 summarizes data for the newborns at risk of developing HBFN, i.e., newborns whose mothers had a positive antibody titer (by the tube method ≥1). Of total six women with anti-D antibody positive in tube method in titer ≥1, two newborns did not have HDFN symptoms, two need phototherapy, but they remained in the nursery besides their mothers while receiving phototherapy and were not admitted to the NICU. Two newborns required blood transfusion. First case (case 12 in tables), a male infant (35+5 weeks, 2.34 kg) was delivered via emergency C-section following a rise in maternal anti-D titers from 32 to 64. Despite a stable birth, the patient (B Rh+, DAT 4+) experienced mild hemolysis and late-onset sepsis, leading to a 3-week decline in hemoglobin (151–105 g/L). He was treated with continuous phototherapy and a top-up transfusion of B-negative RBCs at 20 days of age. The infant was hospitalized for 29 days and was discharged with normalized laboratory parameters. In second case (case 1 in tables), a female infant (38+5 weeks, 3.53 kg) was born via vaginal delivery after maternal antibodies were detected at 34 weeks (titer 16). The infant (O RhD+, DAT 4+) exhibited mild hemolysis, with bilirubin peaking at 284 µmol/L on day 5, requiring continuous phototherapy. Although initially discharged in good health after 9 days, ongoing low-grade hemolysis without significant jaundice necessitated two subsequent top-up transfusions at 20 and 40 days of age. There were no signs of acute bilirubin encephalopathy in the studied group. The frequency of severe HDFN caused by anti-D antibodies undetected at first trimester was in 1.8/10,000.

Table 1.

Pregnancies with late alloimunization and negative antibody screening in the first trimester and newborns from those pregnancies

Immunized pregnant women										Newborns
​	antibodies	blood group	gravidity	parity	previous child	antibody detected	titer at first detection*	titer before delivery*	GA at delivery, weeks#	blood group of child	antigens of child	DAT cord blood
Case 1	Anti-D	B-	1	1	0	8th month	16	16	38	O+	D+	4+
Case 2	Anti-D	AB-	1	1	0	8th month	Neg	8	39	A+	D+	3+
Case 3	Anti-D	A-	1	1	0	8th month	Neg	4	41	A+	D+	2+
Case 4	Anti-D	O-	1	1	0	8th month	Neg	Neg	38	O+	D+	1+
Case 8	Anti-D	A-	1	1	0	8th month	Neg	Neg	40	A+	D+	Neg
Case 5	Anti-D	O-	1	1	0	At birth	NT	NT	​	o+	D+	1+
Case 6	Anti-D	A-	1	1	0	At birth	NT	NT	​	A+	D+	1+
Case 7	Anti-D+C	A-	1	1	0	At birth	NT	NT	​	A+	D+, C+	2/3+
Case 9	Anti-D	AB-	1	1	0	At birth	NT	NT	​	B+	D+	Neg
Case 10	Anti-D	A-	1	1	0	At birth	NT	NT	​	A+	D+	Neg
Case 11	Anti-D	AB-	1	1	0	At birth	NT	NT	​	A+	D+	Neg
Case 12	Anti-D	B-	2	1	0	8th month	16	32	37	B+	D+	4+
Case 13	Anti-D	B-	2	2	Pos	8th month	2	8	39	O+	D+	4+
Case 14	Anti-D	O-	2	2	Pos	8th month	1	4	39	O+	D+	3+
Case 15	Anti-D	O-	2	2	Pos	8th month	Neg	Neg	39	NT	D+	Neg
Case 16	Anti-D	A-	3	3	Pos	8th month	Neg	Neg	40	O+	D+	1+
Case 17	Anti-D	O-	2	1	0	At birth	NT	NT	​	B+	D+	1+
Case 18	Anti-D	B-	2	2	Neg	At birth	NT	NT	​	O+	D+	2/3+
Case 19	Anti-D	O-	2	2	Pos	At birth	NT	NT	​	A+	D+	2+
Case 20	Anti-D	A-	2	2	Pos	At birth	NT	NT	​	A+	D+	Neg
Case 21	Anti-D	A-	2	2	Neg	At birth	NT	NT	​	O+	D+	Neg
Case 22	Anti-D	A-	2	2	Pos	At birth	NT	NT	​	O+	D+	Neg
Case 23	Anti-D	A-	2	1	0	At birth	NT	NT	​	NT	D+	Neg
Case 24	Anti-D+E	A-	2	2	Neg	At birth	NT	NT	​	A+	D+, E+	Neg
Case 25	Anti-D	O-	3	3	Pos	At birth	NT	NT	​	NT	D+	Neg
Case 26	Anti-D	B-	3	2	Pos	At birth	NT	NT	​	B+	D+	Neg
Case 27	Anti-D	AB-	3	3	Pos	At birth	NT	NT	​	AB+	D+	Neg
Case 28	Anti-D+E	O-	4	3	Pos	At birth	NT	NT	​	A+	D+, E+	2+
Case 29	Anti-D	A-	4	2	Pos	At birth	NT	NT	​	NT	D+	Neg
Case 30	Anti-D+C	B-	4	4	Pos	At birth	NT	NT	​	AB+	D+, C+	Neg
Case 31	Anti-D	O-	4	3	Pos	At birth	NT	NT	​	O+	D+	Neg
Case 32	Anti-D+Cw	A-	5	4	Pos	At birth	NT	NT	​	A+	D+, Cw+	Neg
Case 33	Anti-c	O+	2	2	NT	8th month	Neg	4	40	O+	c+	4+
Case 34	Anti-c	A+	3	3	NT	8th month	Neg	1	41	A+	c+	2+
Case 35	Anti-c	B+	3	3	NT	8th month	Neg	1	39	B+	c+	3+
Case 36	Anti-c+E	O+	1	1	NT	8th month	Neg	Neg	39	A+	c+, E+	Neg
Case 37	Anti-c	AB+	2	2	NT	8th month	Neg	Neg	39	AB+	c+	Neg
Case 38	Anti-C	A+	2	2	NT	8th month	Neg	Neg	40	A+	C+	Neg
Case 39	Anti-E	A+	3	2	NT	8th month	Neg	Neg	40	A+	E+	Neg

*Titer of RBC antibodies was tested if antibodies detected during pregnancy, not tested (NT) if antibodies detected at birth.

^#Gestational age was analyzed when antibodies were detected during pregnancy.

Table 2.

HDFN indicators for newborns whose mothers had antibody titers ≥1

Number	Antibody	Titer before delivery	GA at delivery	Induction of labor	DAT	Hb initial (cord or venous)	Hb follow-up	TSB (highest)	Phototherapy, days	IUT or ET	BT, days	NICU admission	Days of stay
Case 1	Anti-D	8	38	No	4+	154	124	284	6	–	20	+	9
Case 2	Anti-D	8	39	No	3+	155	138	196	2	–	–	–	7
Case 3	Anti-D	4	41	No	2+	157	152	111	–	–	–	–	5
Case 12	Anti-D	64	36	Yes	4+	151	105	188	2	–	20	+	29
Case 13	Anti-D	8	39	No	3+	141	125	203	2	–	–	–	6
Case 14	Anti-D	4	39	No	3+	187	–	135	–	–	–	–	4
Case 33	Anti-c	4	40	No	4+	181	163	204	2	–	–	–	5
Case 34	Anti-c	1	41	No	2+	157	–	226	3	–	–	–	5
Case 35	Anti-c	1	39	No	3+	213	199	214	2	–	–	–	5

TSB, total serum bilirubin; IUT, intrauterine transfusion; ET, exchange transfusion; BT, simple blood transfusion.

Regarding non-anti-D antibodies, they have been detected on the repeated screening (34th week of pregnancy) in seven cases. The incidence of non-D immunizations undetected at first-trimester screening but detected during pregnancy was 1/10,000. Anti-c antibodies were the predominant antibodies discovered at repeated screening (5 out of 7 non-anti-D antibodies). Prevalence of Rhc-negative blood donors in our population is 23.4%, and we estimate that of 61,200 tested pregnant women about 14,300 were Rhc-negative pregnant women. The incidence of late anti-c alloimunization in Rhc-negative women was 3.5/10,000. In all cases with late non-D immunization children were antigen-positive, 3 of them had a positive DAT because of anti-c antibodies, and those mothers had positive titer in tube method. One case of anti-C antibody and one case of anti-E were detected on a repeated screening in pregnancy. These neonates were antigen-positive, but their DAT was negative (shown in Table 1). Neonates with positive DAT (N = 3) were monitored for signs of jaundice and anemia. They displayed a mild type of jaundice that required intermittent phototherapy as a single therapeutic measure and were discharged after a short period of hospital stay (shown in Table 2).

Discussion

In this study, we performed RBC antibody screening in 61,200 pregnant women of which 11,000 were RhD-negative and found that anti-D antibodies undetected at first-trimester screening caused severe HDFN in two cases. In previous retrospective study, more than one third of anti-D antibodies with postnatal type Rh immunoprophylaxis were first detected on the repeated screening in pregnancies (28th or 34th week) or at the delivery but were undetected at first-trimester screening [2]. Similar results were confirmed in this prospective study, which means that more than one third of detected anti-D antibodies were the result of FMH during the same pregnancy. Thus, we confirmed the results of the previous retrospective study that anti-D antibodies detected on the repeated screening at about 30 weeks of pregnancy can cause severe HDFN [2]. In this study, the frequency severe HDFN caused by anti-D antibody which was undetected at first-trimester screening was 1.8 to 10,000 RhD-negative women, while in the historical control, the frequency was even 5.1/10,000 [2]. Our literature review did not find any studies which reported the incidence of HDFN due to anti-D antibodies which were first detected on the repeated screening in the same pregnancy.

In the study period, the incidence of severe HDFN that required transfusion treatment in general has decreased compared to historical control [13], so as expected, the incidence of severe HDFN caused by antibodies undetected at first-trimester screening has decreased. Authors found that lower incidence of severe HDFN cured by transfusion is partly due to lower incidence of anti-D and non-D antibodies [13] but largely due to the modified criteria for transfusion treatment of HDFN [5–7, 14–16]. The criteria for transfusion management of HDFN (ETs and simple BTs) have become more restrictive in the newer recommendations [5, 14–16]. Our institution has adopted those restrictive transfusion criteria that have proven beneficial and safe not only to stable term newborns but also to premature newborns of very and extremely low birth weight [14, 15]. Neonates with severe, life-threatening anemia (hydrops fetalis) may present with shock at birth and may require immediate ET [6]. However, in cases of early disease that is not life-threatening, treatment is based on the severity of anemia and hyperbilirubinemia [6, 7]. Thresholds for ET are hematocrit level of less than 25% on the first day of life and hyperbilirubinemia, i.e., bilirubin level above the hour-specific threshold according to the guidelines of the American Academy of Pediatrics (AAP) [6, 7]. HDFN-induced hyperbilirubinemia is managed in the same way as standard neonatal hyperbilirubinemia, following hour-specific algorithms for newborn infants with risk of hemolysis [7]. Other studies report a reduced incidence of severe HBFN treated by ETs [16].

Several studies have found that FMH during pregnancy can cause a negative first-trimester screening test but become positive due to non-D antibodies [2–4]. Some studies found that even 25% of all severe HDFN caused by non-D antibodies were caused by antibodies undetected at first-trimester screening, and most were caused by anti-c antibody [2, 3]. Thus, the authors recommend repeated screening in 30th week of pregnancy, but only for Rhc antigen-negative pregnant women [3]. The results of such practice were analyzed by Slotweg et al. [4] The authors found out of the 62,000 Rhc-negative women tested at 30th week of gestation, 65 anti-c antibodies were first time detected at third semester (incidence 10.5 per 10,000). Severe HDFN requiring transfusion treatment was recorded in two cases [4]. The incidence of late alloimmunization in Rhc-negative women was higher than in all RhD-positive women, as anti-c and anti-E, the most frequent newly detected antibodies in all studies, were found, especially in Rhc-negative women [1–4]. We estimated that there were 14,000 Rhc-negative pregnant women included in this study. The significantly lower number of tested Rhc-negative pregnant women may explain the absence of severe HDFN caused by anti-c or other non-anti-D antibodies resulting from late alloimmunization in this study. Nevertheless, the study documented a lower incidence of late anti-c alloimmunization among Rhc-negative women, at 3.5 per 10,000, compared to the study by Slotweg et al. [4], where the incidence was 10.5 per 10,000. The frequency of anti-c antibodies detected at first-trimester screening and late in pregnancy, according to the results of several studies, varies from 29 to 100 cases per 100,000 pregnant women [3, 17, 18]. The reasons are certainly multiple, differences in the frequency of Rhc-negative individuals in the population, selection of cE-matched RBC products for female transfusion recipients in the generative period, the average number of pregnancies, the sensitivity of the RBC antibody screening test. The sensitivity of methods for antibody screening which we used during this study has increased, causing low-titer non-D antibodies to be detected in the first trimester of pregnancy. In this study, the IAT was performed in automated analyzers based on column agglutination technique with three-cell panel, while in the previous period, the IAT was performed in a tube method or on column agglutination technique with two-cell panel. It is possible that it could be an additional factor that all non-anti-D antibodies that caused severe HDFN were detected in the first trimester.

Some studies found that non-anti-D antibodies first detected in the third trimester caused severe HDFN in multiparous women [2, 4]. This finding could be explained by the fact that women were immunized in previous pregnancies, but antibody titer was below the sensitivity limit of the test. More sensitive methods for RBC antibody screening will be able to detect RBC antibodies that are in low titer [9].

Screening for RBC antibodies in RhD-positive pregnant women during the third trimester has been standard practice in most countries for several decades. It is essential to consider the changes that have occurred over time, including the decreased incidence of HDFN, the increased sensitivity of RBC antibody screening tests, and the fact that, in this study, not a single case of HDFN due to late immunization was observed among more than 60,000 pregnant women tested. Screening for RBC antibodies in Rhc-negative pregnant women with two or more pregnancies would likely be more effective, as this approach would target only 15–20% of RhD-positive pregnant women. The findings of this study may be valuable in informing decisions regarding the necessity of third-trimester antibody screening in RhD-positive women.

Conclusion

Sensitive methods for testing of anti-erythrocyte antibodies, careful monitoring of immunized pregnant women and careful care of newborns, as well as current revised guidelines for the treatment of HDFN are the reasons why it is very likely that a first-trimester anti-erythrocyte antibody screening test will detect all cases of non-anti-D antibodies that could lead to severe HDFN in that pregnancy.

Acknowledgments

The authors are grateful to Jasminka Resic Karara for comments and advice during preparation of the manuscript.

Statement of Ethics

The study protocol was reviewed and approved by the Ethics Committee of the University Hospital of Split (Ethical Approval No. 520-03/24-01/109). Written informed consent was not required according to the decision of the Ethics Committee of the University Hospital of Split.

Conflict of Interest Statement

The authors have no conflicts of interest to declare.

Funding Sources

This study was not supported by any sponsor or funder.

Author Contributions

Dajak Slavica, MD: conceptualization, data curation, formal analysis, investigation, methodology, validation, and writing – original draft. Anet Papazovska Cherepnalkovski, MD: conceptualization, data curation, formal analysis, investigation, and writing – original draft. Paula Odak: data curation, formal analysis, investigation, and writing – original draft.

Funding Statement

This study was not supported by any sponsor or funder.

Data Availability Statement

All data generated or analyzed during this study are included in this article. Further inquiries can be directed to the corresponding author.