Prenatal Blood Typing With Next Generation Sequencing (NGS) - an Implementation Study in HDFN (PREFAB) (PREFAB)

Title

Prenatal blood typing using next-generation sequencing (NGS): an implementation study of red cell alloimmunization in pregnancy (the PREFAB study)

State of the art

Current guidelines recommend prenatal fetal red cell antigen testing for the D, c and K antigens using real-time PCR analysis of cell-free fetal DNA (cffDNA). However, real-time PCR is not available for fetal antigen testing in pregnancies complicated by alloimmunisation against most other clinically relevant red cell antigens. Consequently, fetal antigen status is currently inferred indirectly through maternal and paternal antigen phenotyping.

At birth, approximately 30% of alloimmunised pregnancies are found to involve neonates who are negative for the corresponding red cell antigen on cord blood testing, indicating that these pregnancies were not at risk of fetal haemolytic disease. Cell-free fetal DNA is detectable in maternal plasma early in pregnancy, with reliable concentrations from approximately gestational week 10 onwards, and the fetal DNA fraction increases progressively throughout gestation.

Next-generation sequencing (NGS) has emerged as a transformative technology in prenatal diagnostics, enabling high-throughput genotyping capable of detecting a broad spectrum of genetic variants within a single assay.

Our research group recently completed a method validation study evaluating a novel NGS-based assay for the detection of fetal red cell antigens using cell-free fetal DNA isolated from maternal plasma. Fetal DNA was successfully detected in 97% of pregnancies between gestational weeks 13 and 27 using selected genetic markers. Prenatal fetal antigen typing by NGS demonstrated 100% concordance with postnatal cord blood genotyping.

These findings provide strong proof of concept for the clinical application of this technology. The next step is to evaluate the implementation of this novel NGS-based approach in routine clinical practice through the prospective PREFAB study.

Significance and scientific novelty

The proposed project is the first prospective nationwide study to evaluate the clinical implementation of comprehensive NGS-based fetal antigen typing in early pregnancy. Its scientific novelty lies in replacing conventional single-antigen PCR-based diagnostics with comprehensive genomic fetal antigen typing, thereby enabling accurate identification of pregnancies in which the fetus is not at risk despite maternal alloimmunisation. This approach has the potential to identify an additional 30% of pregnancies that can safely avoid unnecessary fetal surveillance.

The project will evaluate not only the analytical performance of the NGS-based method but also its impact on clinical workflow, patient safety, and health-economic outcomes.

Specific aims

The specific aims of the PREFAB study are to:

Implement prenatal fetal genotyping using next-generation sequencing (NGS) during the first or early second trimester in alloimmunised pregnancies across the seven Swedish university healthcare regions.

Evaluate the impact of NGS implementation on the clinical management of red cell alloimmunisation in pregnancy, including clinical workflow, patient safety, and translational logistics.

Assess the health-economic impact of implementing NGS-based fetal antigen typing compared with conventional clinical management of red cell alloimmunisation during pregnancy.

Project description

Theory and methods

The project comprises a prospective implementation study and a complementary health-economic evaluation. Although NGS has traditionally been regarded as costly and analytically demanding, the development of targeted sequencing assays for specific clinical applications has increasingly enabled its integration into routine diagnostic practice. In parallel, the establishment of an efficient workflow for sample collection, transport, sequencing, analysis, and reporting will be essential to ensure scalability, timely turnaround, and cost-effectiveness in routine clinical care.

Study population and study workflow

Pregnant individuals with confirmed red cell alloimmunisation will constitute the study population. Pregnancies from all seven Swedish university healthcare regions will be enrolled between the second quarter (Q2) of 2026 and the fourth quarter (Q4) of 2028. Individuals with red cell alloimmunisation are identified through the national antenatal screening programme and referred to regional specialist obstetric units according to current clinical practice.

At the first specialist consultation, or during an initial digital consultation, between gestational weeks 8 and 14, eligible participants will receive information about the implementation study and the use of NGS-based fetal red cell antigen typing performed on cell-free fetal DNA (cffDNA) isolated from maternal plasma, corresponding to the maternal red cell antibody identified during routine screening.

A 7 mL maternal blood sample will be collected locally and forwarded to the Department of Clinical Immunology and Transfusion Medicine at Karolinska University Hospital for analysis. Samples will be analysed in batches every two to three weeks, and results will be reported to the responsible clinicians and the participant approximately three weeks after sample collection.

If the initial sample is obtained before gestational week 10, a confirmatory analysis will be performed at gestational week 14 to minimise the risk of a false-negative result due to an insufficient fetal DNA fraction. This follows the current clinical protocol for fetal RhD genotyping by real-time PCR, which will remain unchanged throughout the study. Gestational age may not yet have been confirmed at the time of sampling. In Sweden, gestational dating ultrasound is routinely performed at 12-13 weeks of pregnancy, and the confirmed gestational age may therefore be lower than initially estimated.

Subsequent clinical management will be guided by the NGS result.

Pregnancies in which the fetus is predicted to be negative for the relevant red cell antigen corresponding to the maternal antibody will not undergo fetal surveillance for alloimmunisation. For pregnancies with maternal anti-D, anti-c or anti-K antibodies identified during first-trimester screening, a second NGS analysis will be performed when the fetus is predicted to be antigen-negative as an additional patient safety measure. Routine maternal antibody screening at approximately gestational week 28 will continue according to regional clinical practice.

Pregnancies in which the fetus is predicted to be antigen-positive will be managed according to existing clinical guidelines for red cell alloimmunisation.

At delivery, routine cord blood samples will be collected from all newborns and analysed locally by conventional blood group serology. This will include newborns predicted by NGS to be negative for the relevant antigen. The principal investigator at each study site will verify concordance between the prenatal NGS result and the postnatal cord blood result within five days of delivery. In cases of discordance, an umbilical cord blood sample will be forwarded to the central study laboratory at the Department of Clinical Immunology and Transfusion Medicine, Karolinska University Hospital, Huddinge, within seven days for repeat analysis. The central study coordinator will be notified immediately. Cord blood samples are routinely stored for seven days at all participating laboratories. If a cord blood sample is unavailable, a neonatal capillary blood sample will be collected instead. Neonatal outcomes will be recorded as part of the patient safety evaluation (see separate variable list).

Current clinical management of red cell alloimmunisation during pregnancy is based on paternal red cell antigen phenotyping and serial maternal antibody titre measurements. Pregnancies are not routinely monitored if the corresponding paternal antigen phenotype is negative.

Secondary study component

In the Stockholm region, routine paternal antigen testing will continue throughout the study to determine the proportion of fathers with heterozygous or antigen-negative phenotypes. These data will contribute to the planned health-economic evaluation. Paternal antigen testing is currently part of routine care in the Stockholm region and is coordinated through antenatal care clinics before referral to the specialist obstetric unit at Karolinska University Hospital, Huddinge. In the remaining participating regions, paternal antigen testing will not be performed during the study unless clinically indicated.

Study sites

The study will be conducted in all seven Swedish university healthcare regions: Umeå University Hospital, Uppsala University Hospital, Karolinska University Hospital, Örebro University Hospital, Linköping University Hospital, Sahlgrenska University Hospital, and Skåne University Hospital.

Statistics

Descriptive statistics will be used to summarise baseline demographic and clinical characteristics, sampling parameters, and implementation outcomes. The diagnostic performance of prenatal NGS-based fetal antigen typing will be evaluated against current clinical practice, with and without paternal antigen testing, using sensitivity, specificity, positive and negative predictive values, and corresponding 95% confidence intervals.

Concordance between prenatal NGS-based fetal antigen typing and postnatal cord blood typing will be assessed using measures of diagnostic agreement. Comparisons between clinical management strategies will be performed using appropriate statistical methods according to the distribution and type of data. The health-economic evaluation will compare healthcare costs per pregnancy between the different management strategies using standard health-economic cost-analysis methods.

Calculated study population

Approximately 1% of pregnant individuals in Sweden are diagnosed with clinically relevant red cell alloimmunisation during pregnancy. Assuming participation from all seven Swedish university healthcare regions approximately 700-750 pregnancies are expected to be enrolled over a two-year study period. Allowing for an estimated 25% attrition rate the anticipated evaluable study population will provide a robust basis for the planned study.

Participation from all study regions is expected to be high, supported by the established national collaboration within the Swedish Working Group for Alloimmunisation in Pregnancy (GRAVIMM-ARG), which has facilitated the development and implementation of national clinical recommendations in this field.

Data collection

All study data will be entered into the GravIMM database (www.gravimm.se), an established national database for pregnancies complicated by red cell alloimmunisation. Access to the database will be provided to all participating study sites.

Prospectively collected data will include maternal characteristics, prenatal investigations, ultrasound findings, laboratory results, pregnancy management, delivery details, neonatal outcomes, and other study variables specified in the protocol. Data will be entered by participating investigators and research staff using information obtained from the participants' electronic medical records and accredited laboratory information systems.

All data collection will be performed in accordance with the study protocol and applicable regulations governing data protection and clinical research.

Outcomes

The primary outcome is the successful and safe clinical implementation of prenatal fetal antigen genotyping by next-generation sequencing (NGS) during the first or early second trimester of pregnancy. This will be evaluated through assessment of diagnostic concordance with postnatal cord blood typing, implementation feasibility, patient safety, and predefined implementation outcomes.

The secondary outcome is to compare the diagnostic accuracy and clinical impact of NGS-based fetal antigen typing with current standard clinical management, including conventional fetal antigen assessment based on paternal antigen testing, in the Stockholm cohort.

Safety assessment and adverse events

Discordance between prenatal NGS results and cord blood typing

The principal investigator (PI) at each participating study site is responsible for verifying concordance between the prenatal NGS-based fetal antigen result and the postnatal cord blood typing result within five days of delivery. In the event of discordance, the central study team will be notified immediately. The stored umbilical cord blood sample will be forwarded to the central study laboratory at the Department of Clinical Immunology and Transfusion Medicine, Karolinska University Hospital, Huddinge, within seven days for repeat analysis. Cord blood samples are routinely stored for seven days at all participating laboratories.

Adverse event definitions

If discordance between the prenatal NGS result and cord blood typing is identified but the newborn remains clinically unaffected, the event will be classified as an adverse event (AE).

If discordance is associated with clinically significant neonatal disease, including hyperbilirubinaemia or anaemia requiring treatment, the event will be classified as a serious adverse event (SAE).

Failure to obtain a cord blood sample at delivery will be documented as a protocol deviation (note to file). In such cases, a capillary blood sample will be obtained from the newborn for confirmatory testing.

A turnaround time exceeding four weeks from maternal sample collection to reporting of the NGS result to the responsible clinician will be recorded as an adverse event.

A transport time exceeding five days between maternal blood sampling and receipt of the sample at the central study laboratory will be recorded as an adverse event.

Sub-study: Health-economic evaluation of prenatal fetal red cell antigen typing using next-generation sequencing (NGS)

Objective

To compare the healthcare costs associated with NGS-based prenatal fetal red cell antigen typing with those of current standard clinical management of red cell alloimmunisation during pregnancy.

Study design

Prospective health-economic evaluation conducted alongside the implementation study.

Study setting and participants

The health-economic evaluation will include all pregnancies enrolled in the main study. Healthcare costs will be estimated and compared for three alternative management strategies:

1. conventional clinical management including paternal antigen testing;
2. conventional clinical management without paternal antigen testing; and
3. prenatal fetal red cell antigen typing using NGS.

Outcome measures

The primary outcome will be the mean healthcare cost per alloimmunised pregnancy for each management strategy. Secondary outcomes will include the incremental costs associated with NGS implementation compared with conventional clinical management and the potential cost savings resulting.