NIPT Massively Parallel Sequencing (MPS) for Clinically Significant Fetal Antigens

Frequently asked questions

October 2024

What is NIPT sequencing? 

Non-invasive prenatal testing (NIPT) for clinically significant fetal antigens is a molecular blood group genotyping sequencing assay under development to predict the blood group phenotype of the fetus. This is a current research study looking to develop a massively parallel sequencing (MPS) assay to predict a panel of clinically significant fetal antigens relevant to transfusion from cell free DNA. The advantage of NIPT MPS is its ability to provide fetal genotyping using an extended panel of clinically significant red blood cell (RBC) antigen/ human platelet antigen (HPA), along with the inclusion of internal fetal control markers, in a single, non-invasive test system from 12 weeks gestation. It should be noted that this testing is not yet accredited, and as such results obtained are for research use only.

It is performed for cases where the mother is antigen negative and has a corresponding allo-antibody placing the fetus at risk of being affected by Haemolytic Disease of the Fetus and Newborn (HDFN), or if there is concern for FNAIT and fetal prediction could be helpful. NIPT utilises maternal peripheral whole blood samples. Cell-free DNA (cfDNA), which contains cell-free fetal DNA (cffDNA) is extracted from maternal plasma. The DNA is analysed for the presence of the gene or molecular variation responsible for (or associated with) the blood or platelet group phenotype under investigation.

What do we test for?

The testing is performed using a custom QIAGEN panel, which has selected common antigens for the following blood and platelet groups.  Please be aware that the panel does not include all antigens in the listed blood groups, and not all HPA antigens are covered. 

Red blood cell systems	Human platelet antigens
MNS	HPA-1
RH (RHD/RHCE)	HPA-2
LU	HPA-3
KEL	HPA-4
FY	HPA-15
JK
DI
CO
GE
VEL
AUG
CTL2
MAM

How do we test?

The assay is performed using a custom QIAGEN QIAseq™ Targeted DNA Panel designed to cover RBC and HPA allelic polymorphisms across 12 blood group systems plus six platelet systems, and fetal control markers. The QIAseq 12-index I kit is used as part of this assay to label each sample with adapters containing sequences for sample barcoding. Paired-end sequencing is then performed on the Illumina MiSeq. Maternal genomic DNA (gDNA) samples are tested in tandem with the cfDNA sample. FASTQ files generated from sequencing the cfDNA and gDNA samples are input to a customised bioinformatic workflow (QIAGEN) using CLC Genomics Workbench software to determine the frequency of fetal antigen signals present. Non-specific background reads, commonly associated with MPS methods, are accounted for by subtracting the maternal gDNA allele frequency from the cfDNA allele frequency, forming the ‘subtractive allele frequency’ (SAF). The SAF is used as the threshold for positive and negative fetal antigen predictions, as detailed below.

Performance

The assay has a positive predictive value (PPV) ≥99.9% and negative predictive value (NPV) ≥ 97.1%. The accuracy based on the study data is 98.5% with a 95% Confidence Interval of 91.9% to 99.9%. A proportion of samples will be reported as inconclusive, as explained below. In this instance a repeat sample will be requested. 

How are the results interpreted?

Final patient cfDNA interpretation takes into account the combination of results obtained for patient cfDNA (blood group allele and housekeeper) and maternal gDNA (blood group allele and housekeeper). 
For more information, see McGowan EC, O’Brien H, Sarri ME, Lopez GH, Daly JJ, Flower RL, et al. Feasibility for non-invasive prenatal fetal blood group and platelet genotyping by massively parallel sequencing: A single test system for multiple atypical red cell, platelet and quality control markers. Br J Haematol. 2023;00:1–12.  https://doi.org/10.1111/bjh.19197

Fetal antigen status result	Criteria
Negative	SAF <1.0%
Positive	SAF >2.5%
Conclusive	SAF >1.0% – <2.5%

A research report will be generated and emailed, via ‘Secure Send’, to the referring clinician. 

How early in the pregnancy can samples be tested?

Gestational age must be at least 12 weeks. The concentration of fetal DNA in the mother’s blood increases with the progression of the pregnancy. Any sample collected before 12 weeks gestation can lead to inconclusive or false negative results and will not be tested. 

Multiple pregnancy?

There is limited data demonstrating the feasibility of NIPT for monozygotic (identical) and/or dizygotic (fraternal) twin pregnancies. We are currently only able to report on cases involving monozygotic pregnancies and not on cases involving dizygotic pregnancies. However, we are happy to accept samples from dizygotic pregnancies as there is limited data available for such scenario and the resulting information is valuable for further developing the capability of this test system. 

Sample requirements

• A minimum of 3 x 6 mL whole blood samples. It is preferred to send samples in 1 X 6 mL EDTA and 
  2 x 6 mL maternal cell stabilising tubes, either Roche or Streck. (Lithium Heparin samples are not suitable.) 
• Roche tubes can be requested from Lifeblood R&D (atypicalNIPT@redcrossblood.org). 
• It is possible however to test a sample sent in 3 x 6 mL EDTA.  
• Sample collection should be planned to ensure they are received for processing within 72 hours of collection. 
• Samples should be stored at 2-8 °C. 
• Samples must be provided with completed ‘Study request form’ with a minimum of 3 identifiers and include the tests requested, referring organisation’s contact details and any relevant clinical information. 
• It is very important to include the requesting clinicians’ email for correspondence and reporting.
• Testing cannot be completed without a signed ‘Consent form’, which is also available online at the same location as the ‘Study request form’.  Please send both the study request form and signed consent form together.
• The identifiers on the sample must match the request form exactly. Samples should be sent packaged with a cold ice-brick to the QLD Research and Development Laboratory, via the address below as soon as possible following collection: 
  
  Research and Development, Attention Catherine Hyland
  Australian Red Cross Lifeblood 
  44 Musk Avenue (delivery via Blamey Street)
  KELVIN GROVE
  QLD 4059
  AUSTRALIA 

What is the testing turnaround time?

Sequencing is performed in batches of 5 samples. This can lead to delays in testing if numbers are limited.  The average turnaround time is 8 weeks. If there is urgency for testing, please contact atypicalNIPT@redcrossblood.org to discuss.

Enquiries

Please direct sample collection and shipment enquiries to the Research and Development team via atypicalNIPT@redcrossblood.org. Please direct all clinical enquiries to the Transfusion Specialist for your state. The contact details are available using this link Contact | Lifeblood